СИСТЕМА НАПРАВЛЕННОЙ ДЕГРАДАЦИИ БЕЛКОВ И EE ПРИМЕНЕНИЕ

Abstract

An endoplasmic reticulum-based chimeric protein construct for targeted protein degradation and the use thereof, wherein the chimeric protein construct is used for treating cancers, viral infection diseases, autoimmune diseases, neurodegenerative diseases, etc. The chimeric protein construct includes a protein binding domain based on an endoplasmic reticulum-associated degradation mechanism and a targeting domain. The protein binding domain of the chimeric protein construct based on the ERAD mechanism can be the transmembrane domain of the endoplasmic reticulum resident protein of a virus 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 proteins of interest, and can be the natural ligand of the target protein, an antibody that specifically recognizes the target protein, or an antigen-binding fragment thereof. The targeting domain of the chimeric protein construct can also be an antigen that can be specifically recognized by an antibody.

Description

A targeted protein degradation system and its application Technical Field

[0001] The present disclosure relates to a targeted protein degradation system and its application, particularly in the treatment of diseases (including but not limited to cancer, viral infectious diseases, autoimmune diseases, and neurodegenerative diseases). Background Art

[0002] Targeted protein removal is desirable for at least the following reasons: 1) determining the function of a protein in basic research; 2) validating therapeutic targets; 3) preventing the effects of pathogenic proteins (e.g., tumor proteins or proteins that cause neurodegeneration); 4) enhancing the degradation of proteins that accumulate during aging; and 5) reducing the side effects of cell therapy (e.g., preventing the expression of immunogenic proteins). However, existing targeted protein removal technologies all have their own inherent technical challenges.

[0003] Gene editing technologies (e.g., CRISPR technology) can target and knock out genes encoding any target protein at the genetic level, but there are off-target problems, low specificity in identifying and cutting desired DNA sites, and are prone to side effects such as chromosomal instability.

[0004] Small interfering RNA (siRNA) can block target protein synthesis at the RNA level. However, siRNA also has potential off-target effects. For example, the sense strand of an siRNA can silence the expression of a homologous gene, leading to off-target effects mediated by the sense strand. Furthermore, small mismatches in the small nucleic acid sequence can also lead to silencing of other genes, causing off-target effects and potentially toxic side effects.

[0005] Protein degradation targeted chimera technology (PROTAC) can degrade target proteins at the protein level. It is usually added to cells or administered to animals or humans as a biochemical reagent to induce intracellular degradation of target proteins. Early PROTACs were based on peptide ligand molecules bound to E3 ligases. However, PROTACs prepared using peptide ligand molecules have low cell permeability and instability, resulting in low degradation efficiency. Although PROTACs using small molecules as E3 ligase ligands have shown significant effectiveness, their membrane permeability and bioavailability are poor.

[0006] Therefore, a new targeted protein degradation system is urgently needed to overcome at least one problem existing in various existing technologies for targeted removal of target proteins.

[0007] Summary of the Invention

[0008] On the one hand, the present application provides a chimeric protein construct (chimeric protein construct) that degrades the target protein by endoplasmic reticulum-associated degradation (ER-associated degradation, ERAD) mechanism, which includes an ERAD mechanism protein binding domain and a targeting domain (targeting domain). In some embodiments, the present application provides a general target protein degradation component that can hijack the ERAD mechanism to prevent the transport of the target protein in the ER and promote its translocation into the cytoplasm, so that the target protein is ubiquitinated and degraded by the proteasome-based UPS mechanism, and / or by the ALP mechanism of the autophagy lysosome, the lysosome endocytoses and degrades the target protein. This design can be directed to any protein (including endogenous proteins or exogenous proteins) to effectively inhibit expression and rapid degradation to achieve therapeutic purposes. The present application targets various endogenous target proteins or exogenous target proteins by redesigning viral elements to achieve retention and directional degradation of target proteins in the endoplasmic reticulum.

[0009] On the other hand, the present application provides a chimeric protein construct that synergistically degrades a target protein through the ERAD mechanism and the ubiquitination mechanism, which comprises an ERAD mechanism protein binding domain, a targeting domain, and further a protein degradation pathway member binding domain. This design can greatly improve the degradation efficiency of the target protein and is applicable to many targets that cannot be degraded by PROTAC technology.

[0010] The above two chimeric protein constructs are also referred to as TPD (Targeted Protein Degradation) chimeric protein constructs in this application. The application also provides nucleic acids encoding the above chimeric protein constructs, nucleic acid vectors, oncolytic viruses, and cells expressing the above chimeric protein constructs, and their use in treating diseases.

[0011] Embodiment 1. A chimeric protein construct comprising an endoplasmic reticulum-associated degradation (ERAD) machinery protein binding domain and a targeting domain.

[0012] Embodiment 2. A chimeric protein construct as described in embodiment 1, wherein the ERAD machinery protein binding domain comprises the transmembrane domain of a viral endoplasmic reticulum resident protein or a functional variant thereof and the endoplasmic reticulum resident domain or a functional variant thereof.

[0013] Embodiment 3. The chimeric protein construct of embodiment 1 or 2, wherein the viral endoplasmic reticulum-resident protein is adenovirus E3-19K.

[0014] Embodiment 4. The chimeric protein construct of embodiment 1 or 2, wherein the viral ER-resident protein is not adenovirus E3-19K.

[0015] Embodiment 5. The chimeric protein construct of embodiment 4, wherein the viral endoplasmic reticulum resident protein is selected from at least one of the following: HCMV glycoprotein 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, Cowpox Virus protein CPXV203.

[0016] Embodiment 6. A chimeric protein construct as described in any of the preceding embodiments, further comprising a protein degradation pathway member (e.g., E3 ubiquitin ligase, proteasome, lysosome) binding domain, optionally, the protein degradation pathway member binding domain is connected to the ERAD machinery protein binding domain.

[0017] Embodiment 7. A chimeric protein construct as described in any of the preceding embodiments, 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, double-chain antibody, three-chain antibody and four-chain antibody) that specifically targets the target protein.

[0018] Embodiment 8. A chimeric protein construct as described in embodiment 7, wherein the target protein is a pathogenic protein, optionally, the pathogenic protein is a tumor-related protein, a virus-related protein, an immune function-related protein (including immunosuppressive proteins and immune-activating proteins), an autoantigen protein, or a neurodegenerative disease-related protein.

[0019] Embodiment 9. The chimeric protein construct of embodiment 8, wherein the autoantigen protein is selected from the group consisting of autoantigens associated with type I diabetes, such as islet cell antigen (ICA), insulin (IAA), glutamic acid decarboxylase 65 (GAD65), and insulinoma antigen-2 (IA-2); autoantigens associated with rheumatoid arthritis (RA), such as citrullinated protein / peptide antibodies, heterogeneous nuclear ribonucleoprotein A2 / B1, aldolase, alpha-enolase, calreticulin, heat-activated protein (HSP60), BiP, PGK1, stress-induced phosphoprotein 1, and FUSE-BP1 / 2; autoantigens associated with systemic lupus erythematosus (SLE), such as deoxyribonucleoprotein, SmD1 and SmD3, Clq, leukemia anticoagulant (LA), cardiolipin (CL), and β2 glycoprotein I (β2GP1). I), prothrombin (PT) and phosphatidylserine (PS); autoantigens associated with systemic sclerosis (SSc) / scleroderma (SD), such as Scl-70, SSA, Ro52; antigens associated with autoimmune liver disease, such as mitochondrial antigens, Sp100, PML, gp210, p62; autoantigens associated with myasthenia gravis, such as acetylcholine receptor; autoantigens associated with central nervous system autoimmune diseases (limbic encephalitis, encephalomyelitis, cerebellar ataxia), such as voltage-gated potassium channel (VGKC) complex, voltage-gated calcium channel receptor, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor, gamma-aminobutyric acid-B (GABAB) receptors, glycine receptors; autoantigens associated with multiple sclerosis, such as myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG); autoantigens associated with polymyositis (PM) and dermatomyositis (DM), such as Jo-1, Mi-2, PM-Scl, and Ro-52; autoantigens associated with gluten-sensitive enteropathy, such as endomysial antibodies (EMA) and tissue transglutaminase (tTG); autoantigens associated with anti-NMDAR antibody encephalitis, such as N-methyl-D-aspartate receptor; autoantigens associated with neuromyelitis optica (NMO), such as aquaporin 4 (AQP4); autoantigens related to reproduction, such as ovarian antigens and sperm antigens.

[0020] Embodiment 10. The chimeric protein construct of embodiment 9, wherein the tumor-associated protein is encoded by an oncogene selected from the group consisting of: 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, FE S, 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, KIP 2. 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 / S MAD4, 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 ER-α. receptor, AR) and INT-1, optionally, the tumor-associated protein is selected from the following group: Bcl-2 family members (such as Bcl-2, Bcl-xL and Bcl-w), VEGF / VEGFR, PDGFRβ, EGFR, EGFR mutant, IGF-1R, HDACs, HER2, MYC, KRAS, AFP, CEA, CA199, estrogen receptor (estrogen receptor ER-α), androgen receptor (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.

[0021] Embodiment 11. The chimeric protein construct of embodiment 8, wherein the pathogenic protein is a virus-related protein 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 SARS-CoV-2 RNA-dependent RNA polymerase, herpesvirus DNA and RNA polymerase, herpesvirus capsid glycoprotein, CMV DNA polymerase, CMV capsid glycoprotein, RSV surface protein, RSV capsid protein, RSV RNA polymerase, influenza virus RNA polymerase, influenza virus envelope protein, HPV DNA polymerase, and HPV capsid protein.

[0022] Embodiment 12. A chimeric protein construct as described in embodiment 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.), immune stimulatory / co-stimulatory molecules (e.g., CD3, CD80 / 86, CD28, etc.).

[0023] Embodiment 13. The chimeric protein construct of embodiment 7, wherein the target protein is a target protein associated with a nervous system disease, selected from the group consisting of: Tau, amyloid-β (Aβ), α-synuclein, mutant huntingtin (mHTT), α-synuclein, TAR RNA binding protein (TARDBP) and FUS RNA binding protein (FUS).

[0024] Embodiment 14. The chimeric protein construct of embodiment 2, 4 or 5, wherein the targeting domain does not specifically bind to a TCR.

[0025] Embodiment 15. The chimeric protein construct of embodiment 14, wherein the targeting domain specifically binds to MHC I, MHC II, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and / or ULBP6; preferably, MHC I is HLA I; preferably, MHC II is HLA II.

[0026] Embodiment 16. The chimeric protein construct of embodiment 4, wherein the targeting domain specifically binds to TCR.

[0027] Embodiment 17. A chimeric protein construct as described in embodiment 16, wherein the viral endoplasmic reticulum resident protein is selected from at least one of the following: HCMV glycoprotein 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, Cowpox Virus protein CPXV203.

[0028] Embodiment 18. The chimeric protein construct of any of the preceding embodiments, which is linked to at least one co-expression moiety.

[0029] Embodiment 19. A chimeric protein construct as described in 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 a NK cell activating receptor.

[0030] Embodiment 20. The chimeric protein construct of embodiment 19, wherein the chemokine receptor is selected from the group consisting of: CCR4, CCR5, CCR6, CCR7, CCR9, CCR2b, CXCR1, CXCR2, and CXCR4.

[0031] Embodiment 21. A chimeric protein construct as described in embodiment 20, wherein the NK cell activating receptor comprises: (a) an NK cell activating receptor extracellular domain (ED) or a functional variant thereof, (b) an NK cell activating receptor transmembrane domain (TMD) or a functional variant thereof, and (c) an NK cell activating receptor intracellular domain (ICD) or a functional variant thereof; optionally, a hinge or linker is contained between the NK cell activating receptor extracellular domain or a functional variant thereof, the NK cell activating receptor transmembrane domain or a functional variant thereof and / or the NK cell activating receptor intracellular domain or a functional variant thereof.

[0032] Embodiment 22. A chimeric protein construct as described in 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.

[0033] Embodiment 23. The chimeric protein construct of embodiment 21 or 22, wherein the NK cell activating receptor is complexed with a CNK signaling transduction component.

[0034] Embodiment 24. The chimeric protein construct of any one of embodiments 18-23, wherein the at least one co-expression moiety is linked to the chimeric protein construct via a cleavable linker.

[0035] Embodiment 25. A nucleic acid molecule encoding the chimeric protein construct of any one of embodiments 1-24.

[0036] Embodiment 26. The nucleic acid molecule according to embodiment 25, which is deoxyribonucleic acid (DNA), ribonucleic acid (RNA) (e.g., mRNA, circular RNA, ccRNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with β-D-ribose configuration, α-LNA with α-L-ribose configuration (diastereomers of LNA), 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.

[0037] Embodiment 27. A vector comprising the nucleic acid molecule of embodiment 25 or 26, wherein the nucleic acid molecule is operably linked to at least one polynucleotide regulatory element for expressing the chimeric protein construct encoded by the nucleic acid molecule.

[0038] Embodiment 28. A vector as 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., a transposon DNA) or RNA molecule.

[0039] Embodiment 29. The vector of embodiment 28, wherein the vector is a viral vector selected from the group consisting of retroviruses, lentiviral vectors, adenoviruses, parvoviruses (e.g., adeno-associated viruses), adeno-associated virus (AAV) vectors, coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., malarial and Sendai viruses), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus) and poxviruses (e.g., vaccinia virus, fowlpox virus, and canarypox virus), norwalk viruses, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, baculoviruses, and hepatitis viruses, virus-like particles (VLPs).

[0040] Embodiment 30. The vector of embodiment 29, wherein the viral vector is a retroviral vector.

[0041] Embodiment 31. The vector of embodiment 30, wherein the retroviral vector is selected from the group consisting of: avian leukocytoblastoma, mammalian C-type, B-type virus, D-type virus, HTLV-BLV collection, lentivirus, and foamy virus.

[0042] Embodiment 32. The vector of embodiment 29, wherein the viral vector is a lentiviral vector.

[0043] 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.

[0044] Embodiment 34. The vector of any one of embodiments 27-33, further combined with another vector comprising another nucleic acid molecule encoding at least one co-expression moiety.

[0045] Embodiment 35. An engineered cell expressing the chimeric protein construct of any one of embodiments 1-25, or comprising the nucleic acid molecule of embodiment 26, or comprising the vector of any one of embodiments 27-33.

[0046] Embodiment 36. A cell engineered as described in embodiment 35, which expresses the chimeric protein construct as described in any one of embodiments 1-18, and a co-expression moiety.

[0047] Embodiment 37. The engineered cell of embodiment 35 or 36, wherein the cell is an immune cell selected from the group consisting of: 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.

[0048] Embodiment 38. The engineered cells of embodiment 37, wherein the T cells are 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, a group of regulatory T cells, natural killer T cells, γ-δ T cells, cytokine-induced killer (CIK) T cells and tumor infiltrating lymphocytes.

[0049] Embodiment 39. A method of producing an engineered cell as described in any one of embodiments 35-38, comprising introducing a vector as described in any one of embodiments 27-34 into a starting cell under conditions suitable for expression of a nucleic acid molecule as described in embodiment 25 or 26.

[0050] Embodiment 40. The method of embodiment 39, wherein the starting cell is a stem cell or a cell differentiated from a stem cell.

[0051] Embodiment 41. A method as described in embodiment 38, wherein the stem cell is a hematopoietic progenitor cell (e.g., a T cell progenitor cell, an NK cell progenitor cell, a macrophage progenitor cell), a hematopoietic stem cell (HSC), a CD34+ cell, an embryonic cell line stem cell, a mesenchymal stem cell or an iPSC cell.

[0052] Embodiment 42. The method of embodiment 37, wherein the cell is an immune cell.

[0053] Embodiment 43. The method of 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.

[0054] Embodiment 44. A method as described in embodiment 41, wherein the cells are T cells selected from: CD4+T cells, CD8+T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, a group of cells consisting of regulatory T cells, natural killer T cells, γ-δ T cells, cytokine-induced killer (CIK) T cells and tumor infiltrating lymphocytes.

[0055] Embodiment 45. A cell population produced ex vivo by the method of any one of embodiments 39-44.

[0056] Embodiment 46. A pharmaceutical composition or a kit comprising (i) a chimeric protein construct as described in any one of embodiments 1-24, or a nucleic acid molecule as described in embodiment 25 or 26, or a vector as described in any one of embodiments 27-34, or an engineered cell as described in any one of embodiments 35-38, or a cell population as described in embodiment 45, and (ii) a pharmaceutically acceptable medium.

[0057] Embodiment 47. A method for degrading a target protein, comprising delivering the vector of any one of embodiments 27-34 into a cell expressing the target protein.

[0058] Embodiment 48. The method of embodiment 47, wherein the cells are selected from the group consisting of: tumor cells, virally infected cells, neural cells, transplant cells, and immune cells.

[0059] Embodiment 49. The method of embodiment 47 or 48, wherein the delivering comprises delivery via a viral vector (e.g., an oncolytic virus).

[0060] Embodiment 50. The method of embodiment 49, wherein the vector expresses the chimeric protein construct of any one of embodiments 1-24 within the cell.

[0061] Embodiment 51. A method as described in embodiment 50, wherein the chimeric protein construct is capable of simultaneously binding to the target protein and the ERAD machinery protein, thereby degrading the target protein.

[0062] Embodiment 52. A method of treating a condition or disease in a subject in need thereof, comprising:

[0063] A therapeutically effective amount of the pharmaceutical composition of embodiment 46 is administered to the subject.

[0064] Embodiment 53. The method of embodiment 52, wherein the disease comprises various solid tumors and blood tumors, viral infectious diseases, autoimmune diseases and neurological and degenerative diseases (e.g., Alzheimer's disease), metabolic diseases (e.g., diabetes (e.g., type 2 diabetes), lipid metabolism-related diseases (e.g., tumor lipid metabolism, non-alcoholic fatty liver disease), atherosclerosis (AS), tumor glucose metabolism);

[0065] Preferably, the solid tumor is selected from the group consisting of nervous system tumors, head and neck tumors, chest tumors, digestive system tumors, genitourinary system tumors, soft tissue and skin tumors, bone tumors, etc.;

[0066] Preferably, the nervous system tumors include diffuse glioma, diffuse astrocytoma and anaplastic astrocytoma, glioblastoma, oligodendroglioma, oligoastrocytoma, childhood diffuse glioma, other astrocytomas, ependymoma, neuronal and mixed neuronal-glial tumors, medulloblastoma, other embryonal tumors, schwannoma, meningioma, solitary fibrous tumor and hemangiopericytoma, etc.;

[0067] Preferably, head and neck tumors include nasal cavity and paranasal sinus malignancies, nasopharyngeal cancer, oral cancer, laryngeal cancer, salivary gland tumors, intracranial tumors, thyroid cancer, tongue cancer, etc.;

[0068] Preferably, chest tumors include lung cancer, esophageal cancer, cardia cancer, breast cancer, mediastinal tumors, etc.;

[0069] Preferably, digestive system tumors include gastric cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, small intestinal malignancies, etc.;

[0070] Preferably, genitourinary system tumors include renal cancer, prostate cancer, bladder cancer, testicular malignancy, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, etc.;

[0071] Preferably, soft tissue and skin tumors include malignant fibrous histiocytoma, rhabdomyosarcoma, synovial sarcoma, malignant melanoma of the skin, etc.;

[0072] Preferably, bone tumors include osteosarcoma, Ewing's sarcoma, etc.;

[0073] Preferably, the colon cancer is colon adenoma;

[0074] Preferably, the breast cancer is triple-negative breast cancer cells;

[0075] Preferably, the liver cancer is hepatocellular carcinoma;

[0076] Preferably, the disease is a blood tumor selected from leukemia, lymphoma (HL), multiple myeloma (MM), myelodysplastic syndrome (MDS), etc.;

[0077] Preferably, the leukemia is B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute myeloid leukemia, etc.

[0078] Preferably, viral infectious diseases include: 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, slow virus infectious diseases, etc.

[0079] Preferably, respiratory viral diseases include infections caused by rhinovirus, adenovirus, respiratory syncytial virus, parainfluenza virus and coronavirus; influenza; mumps;

[0080] Preferably, gastrointestinal viral diseases include poliomyelitis; Kuksaki virus infection; ECHO virus infection; viral gastroenteritis: including rotavirus gastroenteritis, Norwalk virus gastroenteritis, adenovirus gastroenteritis, astrovirus gastroenteritis, coronavirus gastroenteritis and calicivirus gastroenteritis;

[0081] Preferably, viral liver diseases include hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, Epstein-Barr virus hepatitis, and cytomegalovirus hepatitis;

[0082] Preferably, viral diseases of the skin and mucous membranes include measles, rubella, roseola infantum, varicella and herpes zoster, smallpox, herpes simplex virus infection, rabies and foot-and-mouth disease;

[0083] Preferably, the ocular viral diseases include epidemic keratoconjunctivitis, follicular conjunctivitis and herpes keratoconjunctivitis;

[0084] Preferably, the viral diseases of the central nervous system include Japanese encephalitis, Western equine encephalitis, Eastern equine encephalitis, St. Louis encephalitis, Venezuelan equine encephalitis, Murray Valley encephalitis, California encephalitis, forest encephalitis and lymphocytic choriomeningitis;

[0085] Preferably, lymphocytic viral diseases include infectious mononucleosis, cytomegalovirus infection and acquired immunodeficiency syndrome;

[0086] Preferably, the insect-borne viral diseases include viral hemorrhagic fevers, including epidemic hemorrhagic fever, yellow fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever, Omsk hemorrhagic fever, Marburg disease and Ebola hemorrhagic fever; dengue fever and dengue hemorrhagic fever; West Nile fever; Colorado tick fever; sand fly fever; etc.

[0087] Preferably, the lentiviral infectious diseases include subacute sclerosing panencephalitis, kuru, progressive multifocal leukoencephalopathy and subacute spongiform encephalopathy (corticostriatal degeneration), etc.

[0088] Preferably, the autoimmune disease includes organ-specific autoimmune disease and systemic autoimmune disease;

[0089] Preferably, the organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, etc.;

[0090] Preferably, the systemic autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, etc.;

[0091] Preferably, the neurological diseases include peripheral nerve diseases of the nervous system such as trigeminal neuralgia, facial paralysis, hemifacial spasm, vestibular neuronitis, glossopharyngeal neuralgia, mononeuropathy, brachial plexus neuralgia, multiple mononeuropathy, polyneuropathy, acute inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy;

[0092] Spinal cord diseases such as myelitis, compressive myelopathy, subacute combined degeneration of the spinal cord, syringomyelia, spinal cord vascular disease, spinal arachnoiditis, etc.;

[0093] Cerebrovascular diseases such as transient ischemic attack, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, intracranial venous thrombosis, etc.;

[0094] Central nervous system infectious diseases such as meningitis, encephalitis caused by viral, bacterial, fungal or parasitic infections, and lentiviral encephalitis caused by lentiviral infections;

[0095] Demyelinating diseases of the central nervous system such as multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, leukodystrophy, etc.

[0096] Movement disorders such as Parkinson's disease, chorea, hepatolenticular degeneration, dystonia, essential tremor, tardive dyskinesia, etc.

[0097] epilepsy;

[0098] Headaches such as migraines, tension headaches, cluster headaches, etc.

[0099] Neurodegenerative diseases such as motor neuron disease, Alzheimer's disease, Lewy body dementia, frontotemporal dementia, multiple system atrophy, etc.

[0100] Genetic diseases of the nervous system such as hereditary ataxia, hereditary spastic paraplegia, Charcot-Marie-Tooth disease, neurofibromatosis, tuberous sclerosis, and cerebrofacial angiomatosis;

[0101] Neurological developmental disorders such as congenital hydrocephalus, cerebral palsy, basilar invagination, and cerebellar tonsillar disease;

[0102] Neuromuscular junction and muscle diseases such as myasthenia gravis, periodic paralysis, polymyositis, progressive muscular dystrophy, myotonic myopathy (myotonic dystrophy, myotonia congenita), metabolic myopathy (mitochondrial myopathy, lipid storage myopathy, glycogen storage disease), etc.

[0103] Autonomic nervous system diseases such as Raynaud's disease, erythromelalgia, hemifacial atrophy, systemic autonomic insufficiency, spontaneous hyperhidrosis, progressive lipodystrophy, etc.

[0104] Nervous system tumors such as glioma, lymphoma, meningioma, etc.:

[0105] Paraneoplastic syndromes of the nervous system such as paraneoplastic cerebellar degeneration, paraneoplastic encephalomyelitis, subacute necrotizing myelopathy, subacute motor neuron disease, paraneoplastic sensory neuron disease, etc.

[0106] Preferably, metabolic diseases include diabetes, hyperlipidemia, gout, etc.

[0107] Embodiment 54. A method of stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 46. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1 shows a chimeric protein construct based on endoplasmic reticulum targeted protein degradation technology, wherein Figure 1A is a linear schematic diagram and structural schematic diagram of the basic ER-TPD chimeric protein construct TBD-TMD-ERD; Figure 1B shows the working structural principle of the ER-TPD chimeric protein construct TBD-TMD-ERD.

[0109] Figure 2 shows an ER-TPD chimeric protein construct TBD-TMD-ERD-E3L having an E3 ubiquitin ligase ligand domain, wherein Figure 2A is a linear schematic diagram thereof, Figure 2B is a structural schematic diagram thereof, and Figure 2C shows the working structural principle of the ER-TPD chimeric protein construct TBD-TMD-ERD-E3L.

[0110] Figure 3 shows an ER-TPD chimeric protein construct TBD-TMD-ERD-E2L having an E2 ubiquitin-binding enzyme ligand domain, wherein Figure 3A is a linear schematic diagram thereof, Figure 3B is a structural schematic diagram thereof, and Figure 3C shows the working structural principle of the ER-TPD chimeric protein construct TBD-TMD-ERD-E3L.

[0111] Figure 4 shows an ER-TPD chimeric protein construct TBD-TMD-ERD-LL having a lysosomal ligand domain, wherein Figure 4A is a linear schematic diagram thereof, Figure 4B is a structural schematic diagram thereof, and Figure 4C shows the working structural principle of the ER-TPD chimeric protein construct TBD-TMD-ERD-LL.

[0112] Figure 5 shows an ER-TPD chimeric protein construct TBD-TMD-ERD-E3L-LL having a combination of an E3 ubiquitin ligase ligand and a lysosomal ligand, wherein Figure 5A is a linear schematic diagram thereof, Figure 5B is a structural schematic diagram thereof, and Figure 5C shows the working structural principle of the ER-TPD chimeric protein construct TBD-TMD-ERD-E3L-LL.

[0113] FIG6 shows the TCR-targeted ER-TPD chimeric nucleic acid construct TCR-ER-TPD, wherein FIG6A shows the chimeric nucleic acid construct TCR-TPD1; FIG6B shows the chimeric nucleic acid construct TCR-TPD2.

[0114] FIG7 shows the results of cell phenotype detection of TCR ER-TPD Jurkat cells at 48 h (A), 96 h (B) and day 6 (C).

[0115] Figure 8 shows an expression vector designed using a single lentiviral expression vector and the EF1α promoter, wherein Figure 8A shows a CNK expression vector; Figure 8B shows a chimeric protein construct HLA-ER-TPD; and Figure 8C shows a chimeric protein construct CNK-HLA-ER-TPD.

[0116] FIG9 shows the flow cytometry results of the cell phenotype of 293 cells transfected with the HLA-abc-targeting ER-TPD chimeric nucleic acid construct combined with a CNK expression vector 48 hours later.

[0117] FIG10 shows an ER-TPD chimeric nucleic acid construct targeting CD123, wherein FIG10A shows a CNK expression vector; FIG10B shows a chimeric protein construct HLA-TPD.

[0118] FIG11 shows the phenotypic flow cytometric results of acute myeloid leukemia cell line THP1 cells transfected with a CD123-targeted scFv TPD chimeric nucleic acid construct 48 hours after transfection.

[0119] Figure 12 shows the application of ER-TPD-CD123 CAR-T cells targeting CD123, wherein Figure 12A1 shows CD123 CAR, Figure 12B1 shows the expression of CD123 on activated T cells, and Figure 12C1 shows that CAR-T cells targeting CD123 produce a self-killing effect during activation culture and killing of tumor cells; Figure 12A2 shows a CD123 CAR-T-T2A-CD123 ER-TPD chimeric protein construct, Figure 12B2 shows that the expression of the chimeric protein construct can degrade self-expressed CD123, and Figure 12C2 shows the killing effect on tumor cells after expressing the chimeric protein construct.

[0120] Figure 13 shows the application of ER-TPD chimeric protein constructs targeting autologous antibodies in the treatment of autoimmune diseases, wherein Figure 13A shows that plasma cells in patients with type 1 diabetes express and release insulin antibodies, which bind to insulin and clear insulin from the peripheral blood, resulting in a severe decrease in the patient's insulin usage rate; Figure 13B shows that ER-TPD transposon DNA targeting insulin antibodies is delivered into plasma cells using plasma cell-targeted liposomes, and is integrated and expressed for a long time.

[0121] Figure 14 illustrates an exemplary chimeric fusion protein containing one or more transmembrane and cytoplasmic domains of TCRαβ or HLA molecule binding molecules and viral ER resident proteins. Among them, Figure 14A is a schematic diagram and composition of the UT chimeric multi-domain. The broad structure of a viral ER resident glycoprotein (VEP) consists of a luminal domain (LD), a transmembrane domain (TMD) and a cytoplasmic functional domain; the UT chimeric multi-domain consists of a TCR or HLA binding domain, a hinge / gasket, a VEP transmembrane and a cytoplasmic domain. Figure 14B is a schematic diagram and composition of the UT chimeric multi-domain. The broad structure of different viral ER resident glycoproteins (VEP) consists of different luminal domains (LD), transmembrane domains (TMD) and cytoplasmic functional domains; the UT chimeric multi-domain consists of a TCR or HLA binding domain, a hinge / gasket, and is different from the VEP transmembrane and cytoplasmic domains. Figure 14C is a schematic diagram and composition of the UT chimeric multi-structure. The broad structure of different viral ER-resident glycoproteins (VEPs) consists of different lumenal domains (LDs), transmembrane domains (TMDs) and cytoplasmic functional domains; the UT chimeric multidomain consists of TCR or HLA binding domains, hinges / gaskets, and different VEP transmembrane and chimeric cytoplasmic domains originate from different VEPs.

[0122] Figure 15 shows that forced expression of the UT element can effectively inhibit TCR expression in Jurkat cells. A lentiviral vector with a truncated EGFR was constructed, encoding an anti-TCR scFv fused to different viral ER-resident proteins, HCMV US2, US3, US11, and the adenovirus E19 TM / CT domain, as well as a lentiviral vector encoding an anti-TCR scFv fused to both US2 and adenovirus E19 TM / CT. Jurkat cells were transduced with this lentivirus, and untransduced Jurkat cells were used as a control. 48 hours after transduction, the cells were sent to a flow cytometer to detect TCRαβ expression in the transduced cells (EGFR+ cells). The results showed that all EGFR(+) Jurkats showed a significant downregulation of TCRαβ expression; cells transduced with the combined UT components (US2 / E19) could effectively block TCRαβ expression. All tested UT chimeric elements (US2, US3, US11, E19) could block TCRαβ expression. Combining UT elements (US2 and E19) significantly enhanced the inhibitory effect on TCRαβ expression on the surface of Jurkat cells.

[0123] Figure 16 shows that forced expression of UT elements can effectively inhibit the expression of TCR in human T cells. Among them, Figure 16A shows that forced expression of UT elements can effectively inhibit the expression of TCR in human T cells. The lentiviral vector with an expression cassette including a truncated EGFR encodes an anti-TCR scFv (anti-TCR-US2 TM / CT-T2A-EGFRt) fused to the HCMV US3 TM / CT domain. Human T cells were stimulated and transduced with this lentivirus, and non-transduced T cells were used as a control. After 5 days of transduction, the cells were sent to a flow cytometer to detect the expression of TCRαβ in transduced T cells (EGFR+ cells). The results showed that compared with non-transduced T cells, EGFR(+)T showed a significant decrease in TCRαβ expression. This indicates that the US element containing anti-TCR-US3 TM / CT can 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. The lentiviral vector carrying an expression cassette containing a truncated EGFR encodes an anti-TCR scFv fused to the HCMV E19 TM / CT domain (anti-TCR-E19 TM / CT-T2A-EGFRt). Human T cells were stimulated and transduced with this lentivirus, and untransduced T cells were used as a control. Five days after transduction, the cells were sent to a flow cytometer to detect TCRαβ expression in the transduced T cells (EGFR+ cells). The results showed that EGFR(+) T cells showed a significant downregulation of TCRαβ expression compared to untransduced T cells. Increasing viral transduction can further enhance the inhibition of TCRαβ expression on the T cell surface. This indicates that the US element containing anti-TCR-E19 TM / CT can effectively express TCRαβ in human T cells.

[0124] Figure 17 shows that CNK-T cells with UT elements downregulated TCR expression on CD8 / CD4 cells. The lentiviral vector contains an expression cassette (CNK-UT-E19) encoding a chimeric NK receptor and an anti-TCR scFv fused to the E19 TM / CT domain. Human T cells were transduced with this lentivirus. Five days after transduction, the cells were sent to a flow cytometer to detect TCRαβ expression in transduced cells (NKG2D+ cells), conventional chimeric NK receptor-transduced T cells, and non-transduced T cells. The results showed that the introduction of the UT element into the CNK expression cassette did not affect the expression of the chimeric NK receptor. In addition, it can significantly inhibit the expression of TCR on CD8+ and CD4+CNK-T cells.

[0125] Figure 18 shows that CNK-T cells with UT elements exhibit potent cytotoxicity against tumor cells, just like conventional 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, the cells were harvested and flow cytometry analyzed to detect tumor lysis and T cell activation. The data showed that CNK-UT cells exhibited potent cytotoxicity against HepG2 cells comparable to that of conventional CNK-T cells. After 24 hours of co-culture, the number of CD45(-) tumor cells was significantly reduced in both CNK-T and CNK-UT co-cultures. In addition, both CD8 and CD4 CNK-UT cells upregulated CD25 and CD137 after co-culture with HepG2 cells.

[0126] Figure 19 shows four structures of CNK-UT multifunctional complexes, among which Figure 19A is a basic CNK-UT multifunctional complex; Figure 19B is a CNK-UT multifunctional complex with an additional MHC I-binding protein molecular domain; Figure 19C is a CNK-UT multifunctional complex with an additional MHC I-binding protein molecular domain and a composite adapter; Figure 19D is a CNK-UT multifunctional complex with an additional MHC I-binding protein molecular domain and a receptor such as CAR or TCR that targets and kills tumor cells.

[0127] Figure 20 shows the structures of four CNK-UT elements expressing the CNK-UT multifunctional complex, wherein, as shown in Figure 20A, a single lentiviral EF1α promoter expression vector is used to drive the expression of one of the combinations of CNK-UT elements: DAP10-DAP12 ICD-T2A-NKG2D-p2A-anti-TCR-AdE3 ERAD; as shown in Figure 20B, a single lentiviral EF1α promoter expression vector is used to drive the expression of one of the combinations of CNK-UT elements: DAP10-DAP12 ICD-T2A-NKG2D-p2A-anti-TCR-US2 ERAD; as shown in Figure 20C, a single lentiviral expression vector is used to regulate DAP10-CD3ζ-T2A-NKG2D-p2A-ant-TAA scFv-DAP10 and anti-TCR-AdE3, respectively, using the EF1α and CMV promoters. The expression of ERAD-E2A-AdE3 multiple genes was achieved by expressing multiple CNK-UT functional elements on a single 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. After co-transfection of T cells, multiple CNK-UT functional elements were expressed on the same T cell.

[0128] FIG21 shows the flow cytometric detection results of the basic phenotype of CNK-UT (DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3) cells.

[0129] Figure 22 shows the recognition and specific killing results of CNK-UT cells on the human colon adenoma cell line HT29, wherein Figure 22A shows the expression of different NK target proteins of the human colon adenoma cell line HT29; Figure 22B shows that CNK-UT cells have efficient killing and activation functions on HT29.

[0130] Figure 23 shows the recognition and specific killing of MDA-MB453 by CNK-UT cells, wherein Figure 23A shows the expression of different NK target proteins in the triple-negative breast cancer cell line MDA-MB453; Figure 23B shows that CNK-UT cells exhibit efficient killing and activation functions against THP1.

[0131] Figure 24 shows that GPC3 CAR / CNK-UT cells have more efficient tumor clearance ability in mice than GPC3 CAR-T cells.

[0132] Figure 25 shows that GPC3 CAR / CNK-UT cells have more efficient tumor clearance ability than GPC3 CAR-T. DETAILED DESCRIPTION

[0133] Before describing the present disclosure in more detail, it should be understood that the present disclosure is not limited to the particular embodiments described, as such may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0134] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are now described.

[0135] All publications and patents cited in this specification are incorporated herein by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and are incorporated herein to disclose and describe the methods and / or materials in connection with the cited publications. 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 virtue of prior disclosure. Furthermore, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0136] As will be apparent to those skilled in the art after reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0137] I. Definition

[0138] The following definitions are provided to assist the reader. Unless otherwise defined, all technical terms, symbols, and other scientific or medical terms or expressions used herein are intended to have the meanings commonly understood by those skilled in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a substantial difference from the definition of a term as commonly understood in the art.

[0139] Note that in this disclosure, terms such as "comprises / comprised / comprising," "contains / containing," etc. have the meanings ascribed to them in U.S. patent law; they are inclusive or open-ended and do not exclude additional unrecited elements or method steps. Terms such as "consisting essentially of / consists essentially of" have the meanings ascribed to them in U.S. patent law; they permit the inclusion of additional ingredients or steps that do not materially affect the basic and novel characteristics of the claimed invention. The terms "consists of / consisting of" have the meanings ascribed to them in U.S. patent law; that is, these terms are closed-ended.

[0140] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0141] As used herein, the term "about" refers to a measurable value, such as an amount, duration, etc., and includes variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% from the specified value.

[0142] An "antigen" refers to a molecule that can be specifically recognized and bound by an antibody. In some embodiments, the antigens described herein can be molecules that elicit an immune response. This immune response can be a humoral response, a cell-mediated response, or both. Those skilled in the art will appreciate that any macromolecule, including virtually any protein or peptide, can serve as an antigen. It will be apparent that the antigens disclosed herein include therapeutic antibodies capable of eliciting an immune response.

[0143] "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 comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain (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 conserved regions, called framework regions (FRs). 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, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol., Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature, Dec 21-28;342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991); Marie-Paule et al., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol., 186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature, Dec 21-28;342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991); Marie-Paule et al., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol., 273(4), 927 (1997); Choth 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). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0144] As used herein, the "functional fragment" of an antibody is used interchangeably with "antigen-binding fragment" and refers to an antibody fragment comprising one or more CDRs, or any other antibody portion that binds to an antigen but does not comprise a complete native antibody structure. Examples of antigen-binding fragments include, but are not limited to, bifunctional antibodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), single-chain antibody molecules (scFv), scFv dimers (divalent bifunctional antibodies), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody binds. In certain embodiments, an antigen-binding fragment may comprise one or more CDRs from a specific parent antibody.

[0145] "Fab" with respect to antibodies refers to a monovalent antigen-binding fragment of an antibody, which is composed of a single light chain (variable and constant regions) bound to the variable region and first constant region of a single heavy chain via a disulfide bond. Fab can be obtained by papain digestion of antibodies at residues N-terminal to the disulfide bonds between the heavy chains in the hinge region.

[0146] "Fab'" refers to a Fab fragment that includes a portion of the hinge region and can be obtained by pepsin digestion of an antibody at a residue close to the C-terminal end of the disulfide linkages between the heavy chains of the hinge region and therefore differs from Fab in a few residues in the hinge region, including one or more cysteines.

[0147] "F(ab')2" refers to a dimer of Fab', which is composed of two light chains and portions of two heavy chains.

[0148] "Fv," with respect to antibodies, refers to the smallest antibody fragment with a complete antigen-binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain. "dsFv" refers to a disulfide-stabilized Fv fragment in which the linkage between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.

[0149] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody composed of a light chain variable region and a heavy chain variable region linked directly or via a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)). "scFv dimer" refers to a single chain comprising two heavy chain variable regions and two light chain variable regions with a linker. In certain embodiments, the "scFv dimer" is a bivalent bifunctional antibody or bivalent scFv (BsFv) comprising a VH-VL moiety (linked by a peptide linker) dimerized with another VH-VL moiety, such that the VH of one moiety coordinates with the VL of the other moiety and forms two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, the "scFv dimer" is a bispecific, bifunctional antibody comprising VH1-VL2 (connected by a peptide linker) in combination with VL1-VH2 (also connected by a peptide linker), such that VH1 is coordinated with VL1 and VH2 is coordinated with VL2, and each coordination pair has a different antigenic specificity.

[0150] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of a scFv linked to the Fc region of an antibody.

[0151] "Camelized single domain antibody", "heavy chain antibody", "nanobody" or "HCAb" refers to an antibody containing 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 Camelidae (camels, dromedaries and llamas). Although devoid of light chains, camelized antibodies possess a bona fide antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3;363(6428):446-8 (1993); Nguyen VK. et al., “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation,” Immunogenetics. Apr;54(1):39-47 (2002); Nguyen VK. et al., Immunology. May;109(1):93-101 (2003)). The variable domain (VHH domain) of a heavy chain antibody represents the smallest known antigen-binding unit produced by the adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov;21(13):3490-8. Epub 2007 Jun 15 (2007)). "Bibodies" include small antibody fragments with two antigen-binding sites, wherein the fragment comprises a VH domain linked to a VL domain in a single polypeptide chain (VH-VL or VL-VH) (see, e.g., Holliger P. et al., PNAS Jul 15;90(14):6444-8 (1993); EP 404097; WO 93 / 11161). Because the linker is too short, the two domains on the same chain cannot pair, and therefore, the domains are forced to pair with complementary domains from another chain, thereby generating two antigen-binding sites. The antigen binding sites can target the same or different antigens (or epitopes).

[0152] "Domain antibodies" refer to antibody fragments that contain only the variable region of a heavy chain or the variable region of a light chain. In certain embodiments, two or more VH domains are covalently joined by 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.

[0153] In certain embodiments, "(dsFv)2" comprises three peptide chains: two VH portions connected by a peptide linker, and bound to two VL portions by disulfide bridges.

[0154] In certain embodiments, a "bispecific ds diabody" comprises VH1-VL2 (connected by a peptide linker) bound to VL1-VH2 (also connected by a peptide linker) via a disulfide bridge between VH1 and VL1.

[0155] In certain embodiments, a "bispecific dsFv" or "dsFv-dsFv'" comprises three peptide chains: a VH1-VH2 portion, wherein the heavy chain is bound by a peptide linker (e.g., a long flexible linker) and paired with the VL1 and VL2 portions, respectively, via disulfide bridges. Each disulfide-paired heavy and light chain has a different antigenic specificity.

[0156] "Autologous cells" refer to any cells that originate from the same individual as the recipient of the cells (ie, the recipient themselves).

[0157] "Allogeneic cells" refer to any cells that originate from an individual of the same species as the recipient of the cells (ie, not the recipient themselves).

[0158] As used herein, "construct" and "protein construct" are used interchangeably to refer to a protein or protein complex. The term "complex" refers to an aggregate or assembly formed by the aggregation or assembly of more than one protein (e.g., two, three, four, or more proteins). Preferably, the complex can perform a function that the individual proteins cannot. Within the complex, the constituent proteins may or may not be in contact with each other.

[0159] As used herein, the term "chimeric," when used in relation to a construct, means that one portion of the construct is derived from one species, while another portion of the construct is derived from another species.

[0160] As used herein, the term "vector" refers to a medium into which a polynucleotide encoding a protein can be operably inserted to cause expression of the protein. A vector can be used to transform, transduce, or transfect a host cell so that the genetic elements it carries are expressed in the host cell. 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); bacteriophages, such as lambda phages or M13 phages; and animal viruses. Classes of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). A vector can contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector can contain an origin of replication. A vector can also include materials that facilitate its entry into cells, including but not limited to viral particles, liposomes, or protein coatings. A vector can be an expression vector or a cloning vector. The present disclosure provides vectors (e.g., expression vectors) containing the nucleic acid sequence encoding the 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 selection marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phage and M13 phage, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p1 5TV-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, etc.

[0161] As used herein, the phrase "host cell" refers to a cell into which an exogenous polynucleotide and / or vector has been or is to be introduced.

[0162] As used herein, the term "functional variant" refers to a polypeptide / protein that has at least 70% sequence identity with a parent polypeptide / protein but still retains the function of the parent polypeptide / protein. The variant may differ 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.

[0163] In this application, the percentage of "identity" or "sequence identity" of a polypeptide or polynucleotide is determined by comparing two optimally aligned sequences in a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) so that the two sequences are optimally aligned. The percentage is calculated as follows: the number of positions where the identical nucleic acid base or amino acid residue occurs in the two sequences is determined to give the number of matched positions, the number of matched positions is divided by the total number of positions in the comparison window, and the result is multiplied by 100 to give the percentage of sequence identity. Alignment for the purpose of determining % amino acid (or nucleic acid) sequence identity can be performed, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of the National Center for Biotechnology Information (NCBI), see also Altschul SF et al., Journal of Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin et al., Methods in Enzymology, 267:383-402 (1997)). MA et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007) and ALIGN or Megalign (DNASTAR) software. One skilled in the art can use the default parameters provided by the tools, or can customize parameters suitable for the alignment, for example by selecting an appropriate algorithm. In certain embodiments, residue positions that are not identical can differ by conservative amino acid substitutions. A "conservative amino acid substitution" is a conservative amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution does not substantially change the functional properties of a protein. Where two or more amino acid sequences differ from each other by a conservative substitution, the percentage or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. The manner in which such adjustments are made is well known to one skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference.

[0164] "Operably connected" refers to the functional relationship between two or more polynucleotide sequences. In the context of a polynucleotide encoding a fusion protein (such as the polypeptide chain of a CAR of the present disclosure), the term refers to the joining of two or more polynucleotide sequences so that the two polynucleotides remain in the same expression frame so that they can translate an amino acid sequence. In the context of transcription or translation regulation, the term refers to the functional relationship between a regulatory sequence and a coding sequence, for example, a promoter is in the correct position and direction of the coding sequence to regulate transcription.

[0165] "Polynucleotide" or "nucleic acid" refers to a chain of nucleotides. As used herein, polynucleotides include all polynucleotide sequences obtained by any means available in the art, including but not limited to recombinant and synthetic means.

[0166] "Polypeptide" and "protein" are used interchangeably to refer to a chain of amino acid residues covalently linked by peptide bonds. Polypeptides include naturally occurring peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0167] "T cell receptor" or "TCR" refers to the protein complex on the surface of T cells that is responsible for recognizing antigen fragments as peptides bound to MHC molecules.

[0168] The term "chimeric natural killer receptor" (CNK receptor) refers to a composition comprising a chimeric NK activating receptor component and a chimeric NK signal transduction component.

[0169] The term "CNK T cell" refers to a T cell containing a CNK construct.

[0170] The term "chimeric natural killer receptor-Universal T cell" (CNK-UT) refers to a T cell that expresses an NK activating receptor component construct, a CNK signaling transduction component construct, and a UT component construct.

[0171] The term "CAR / CNK-UT" stands for chimeric antigen receptor and chimeric natural killer receptor universal T cell (CNK-UT), which refers to T cells that express a CAR structure targeting a specific tumor antigen, an NK activation receptor component construct, a CNK signal transduction component construct, and a UT component construct.

[0172] 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 to cognate costimulatory molecules on T cells, thereby providing a signal that mediates T cell responses, including but not limited to proliferation, activation, differentiation, etc., in addition to the primary signal provided by, for example, the binding of the TCR / CD3 complex to the peptide-loaded MHC molecule. Costimulatory ligands may include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory 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 Toll ligand receptors, and ligands that specifically bind to B7-H3. Co-stimulatory ligands also include antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0173] "Costimulatory molecule" or "costimulatory receptor" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating 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 also include non-natural engineered proteins.

[0174] As used herein, a "co-stimulatory signal" refers to a signal that, in conjunction with a primary signal, such as TCR / CD3 ligation, results in T cell proliferation and / or upregulation or downregulation of key molecules.

[0175] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a 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 downregulation of TGF-β, and / or reorganization of cytoskeletal structure, among others.

[0176] As used herein, the term "stimulatory molecule" refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell.

[0177] 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 on a T cell (referred to herein as a "stimulatory molecule"), thereby mediating a primary response of the T cell, including but not limited to activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, among others, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0178] As used herein, "activated" refers to a state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cell" particularly refers to a T cell that is undergoing cell division.

[0179] "Effective amount" or "therapeutically effective amount" refers to an amount of a cell, composition, formulation, or any material described herein that is effective to achieve a desired biological result. Such results may include, but are not limited to, elimination of B cells expressing a specific B cell receptor (BCR) and the antibodies produced thereby.

[0180] As used herein, a "subject" is a mammal, such as a human or other animal, and is typically a human. In some embodiments, the subject, such as a patient, to which the cells, cell populations, or compositions are administered is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or ape. The subject can be male or female and can be of any suitable age, including infants, teenagers, adolescents, adults, and elderly subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

[0181] As used herein, "treat" (and grammatical variations thereof, e.g., "treat" or "treatment") refers to the complete or partial improvement or alleviation of a disease or condition or disorder, or a symptom, adverse effect, outcome, or phenotype associated therewith. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviation of symptoms, alleviation of any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, alleviating or relieving the disease state, and alleviating or improving prognosis. These terms do not imply a complete cure of the disease or the complete elimination of any symptom or the effect on all symptoms or consequences.

[0182] As used herein, "prevention" includes providing protection against the occurrence or recurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease. In some embodiments, provided cells and compositions are used to delay the development of a disease or slow the progression of a disease.

[0183] As used herein, "inhibiting" a function or activity refers to reducing the function or activity when compared to the same conditions (except for the conditions or parameters of interest), or when compared to additional conditions. For example, a cell that inhibits tumor growth reduces the growth rate of the tumor compared to the growth rate of the tumor in the absence of the cell.

[0184] In the context of administration, an "effective amount" of an agent (e.g., a chimeric antigen receptor, polynucleotide, vector, pharmaceutical formulation, cell, or composition) refers to an amount effective, at dosages / amounts, and for periods of time necessary, to achieve the desired result, e.g., a therapeutic or prophylactic result.

[0185] A "therapeutically effective amount" of an agent (e.g., a pharmaceutical formulation or cell) is an amount effective to achieve a desired therapeutic effect (e.g., for treating a disease, condition, or disorder, and / or a pharmacokinetic or pharmacodynamic effect of the treatment) at the necessary dosage and time period. A therapeutically effective amount can vary depending on factors such as the disease state, the age, sex, and weight of the subject, and the cell population being administered. In some embodiments, provided methods comprise administering cells and / or compositions in an effective amount, such as a therapeutically effective amount.

[0186] A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the dosage and time period necessary. Typically, but not necessarily, a prophylactic effective amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease. In cases of lower tumor burden, a prophylactically effective amount will in some aspects be higher than a therapeutically effective amount.

[0187] II. Target Protein Degradation (TPD)

[0188] The present invention provides a novel targeted protein degradation system for targeting specific proteins (i.e., target proteins) to block their expression (e.g., surface expression) and accelerate ubiquitination-mediated degradation through the endoplasmic reticulum-associated degradation (ER-associated degradation, ERAD) mechanism.

[0189] Endoplasmic reticulum-associated protein degradation (ERAD) refers to a cellular pathway that targets misfolded proteins in the endoplasmic reticulum for ubiquitination and subsequent degradation by the proteasome. The ERAD process can be divided into three steps: (1) recognition of misfolded or mutated proteins in the endoplasmic reticulum; (2) reverse transport of the identified misfolded or mutated proteins from the endoplasmic reticulum to the cytoplasm: terminally misfolded proteins must be transported from the endoplasmic reticulum back to the cytoplasm, which contains the ubiquitin-proteasome system (UPS); (3) ubiquitin-dependent degradation of the identified 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).

[0190] Here, the present application provides a new chimeric strategy that fuses a targeting domain (such as a specific protein-targeting antibody fragment (ScFv)) with an ERAD functional motif (such as 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 (for example, the expression of a specific protein on the cell surface).

[0191] The ER-TPD technology platform provided in this application has the following advantages over existing protein degradation / removal technologies (e.g., PROTAC, CRISPR, etc.): 1. The ER-TPD chimeric protein construct is delivered to the cell through a carrier (e.g., liposomes, nanoparticles, lentivirus, adenovirus, oncolytic virus, etc.) for stable expression and function. Compared with PROTAC, there is no problem of low cell permeability and short half-life leading to low degradation efficiency; 2. The ER-TPD technology is based on the ERAD principle of the endoplasmic reticulum, which directly intervenes in protein synthesis and achieves efficient degradation during the synthesis and assembly of endoplasmic reticulum proteins. Therefore, ER-TPD 3. Exogenous PROTAC cannot effectively degrade membrane proteins and secretory proteins, while ER-TPD can directly target and degrade retained cell membrane proteins and secretory proteins during the synthesis and assembly of endoplasmic reticulum proteins. Therefore, ER-TPD can target a large number of important disease-related target proteins that cannot be targeted by conventional TPD technology, providing a new drug development approach for disease treatment. 4. In the design method of ER-TPD, the targeting domain can adopt various single-chain antibody structures or artificial affinity ligand structures, which greatly improves the specific and accurate recognition and binding ability of the target protein. Conventional PROTAC technology uses small molecule chemical structures, which makes it difficult to ensure the specificity of the targeting structure. Therefore, ER-TPD technology has the strong advantages of targeting accuracy and specificity. 5. ER-TPD technology can achieve a multi-pathway mechanism for target protein degradation through downstream degradation-guided ligand structures, including the UPS mechanism based on the ubiquitin proteasome and the ALP mechanism of the autophagy lysosome. The downstream degradation-guided ligand structure design can be flexibly connected to the degradation system in the cell through genetic engineering methods to achieve efficient targeted degradation, while conventional PROTAC technology can only achieve degradation by targeting E3 ligases. 6. ER-TPD technology 7. ER-TPD technology degrades target proteins at the protein level. Compared with technologies that block target protein expression at the gene level (such as CRISPR technology), it does not have toxic side effects such as chromosomal instability caused by off-target effects or gene editing processes; 7. ER-TPD technology can be perfectly combined with gene and cell therapy technologies, greatly expanding the current clinical indications of gene and cell therapy and enhancing clinical effects; 8. The development of targeted degradation drugs based on ER-TPD technology is highly feasible and has a short cycle. Nucleic acid and cell drugs based on ER-TPD technology are easier to achieve large-scale and industrial production, and are an important basic platform technology for the development of new drugs.

[0192] In one aspect, the present disclosure provides a chimeric protein construct of Endoplasmic Reticulum-based Targeted Protein Degradation (ER-TPD) technology, which comprises an endoplasmic reticulum-associated degradation (ERAD) mechanism protein binding domain and a targeting domain.

[0193] In certain embodiments, the chimeric protein construct further comprises a protein degradation pathway member (e.g., a ubiquitin-proteasome system pathway member, an endosome-lysosome pathway member, an autophagy pathway member) binding domain. The protein degradation pathway member binding domain can be linked to the constituent elements of the chimeric protein construct in any suitable manner, for example, to the ERAD machinery protein binding domain.

[0194] In another aspect, the present disclosure also provides a library of compositions, wherein each composition comprises a viral protein or a nucleic acid molecule encoding the same, which comprises a functional motif capable of hijacking the ERAD machinery to arrest and accelerate proteasome-mediated degradation within the ER and an affinity portion that targets a specific protein for degradation, for therapeutic purposes to modify cell phenotype, improve cell function, induce apoptosis, or inhibit viral replication.

[0195] 1. ERAD mechanism protein binding domain

[0196] As used herein, the term "ERAD machinery protein" refers to a protein that participates in the ERAD mechanism 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, for example, a transmembrane domain (or a functional variant thereof) and a cytoplasmic domain (or a functional variant thereof) of a viral endoplasmic reticulum (ER) resident glycoprotein, the cytoplasmic domain also referred to as an "endoplasmic reticulum retention domain" or "ER retention domain".

[0197] 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 target proteins.

[0198] i. ERAD machinery protein binding domain derived from adenovirus E3-19K

[0199] In some embodiments, the viral endoplasmic reticulum-resident protein is adenovirus E3-19K. Adenovirus E3-19K (also known as "E19") contains three functional modules: a lumenal domain for interacting with MHC-I and MICA / B molecules, a transmembrane domain, and an endoplasmic reticulum-resident domain, wherein its endoplasmic reticulum-resident domain contains a dilysine motif in the cytoplasmic tail that can return the Golgi apparatus to the endoplasmic reticulum, and therefore can also be called an "ER return signal motif". Studies have shown that the transmembrane domain and the ER return signal motif are required to ensure effective ER localization, transport inhibition of the histocompatibility complex class I (MHC-I) and MHC-I-related chains A and B (MICA / B molecules), and proteasomal degradation. Therefore, adenovirus E3-19K can reside MHC class I molecules (e.g., MHC-I and MICA / B molecules) in the secretory pathway and interfere with antigen presentation. The chimeric protein construct provided by the present disclosure utilizes the transmembrane domain and endoplasmic reticulum retention domain (ie, ERAD degradation domain) of proviral E3-19K to fuse with the targeting protein binding domain, thereby achieving the purpose of target protein degradation.

[0200] In some preferred embodiments, the ERAD degradation domain of the adenovirus E3-19K comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 60, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity. The ERAD degradation domain of the adenovirus E3-19K has a dilysine motif in the cytoplasmic tail, which is capable of returning the Golgi apparatus to the endoplasmic reticulum. In some preferred embodiments, the amino acid sequence of the ERAD degradation domain of the adenovirus E3-19K is as shown in SEQ ID NO. 60.

[0201] ii. ERAD machinery protein binding domains derived from other viral glycoproteins

[0202] In some embodiments, the viral ER-resident protein is not adenoviral E3-19K.

[0203] In some embodiments, the viral endoplasmic reticulum resident protein is selected from at least one of the following: HCMV glycoprotein 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 glycoprotein US2 and US11.

[0204] In some embodiments, the viral endoplasmic reticulum resident protein comprises E3-19K and at least one selected from the group consisting of HCMV glycoprotein 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.

[0205] The HIV-1 Vpu protein is a protein localized to the ER membrane and targets newly generated CD4 in the ER for degradation by the cytoplasmic proteasome. Vpu retains CD4 in the ER primarily through SCFβ-TrCP-dependent ubiquitination and transmembrane domain (TMD) interactions.

[0206] a) HCMV US2 and US11

[0207] Two HCMV proteins, US2 and US11, are ER-resident type I integral membrane glycoproteins that co-opt the ERAD pathway to promote the degradation of MHC class I heavy chains, thereby inhibiting MHC class I antigen presentation. Expression of either protein results in rapid degradation of newly synthesized MHC class I heavy chains. US2 and US11 bind to MHC class I heavy chains through their luminal domains and recruit host cell proteins that extract the polypeptides from the ER membrane by "pulling" on the cytoplasmic tail of the heavy chain. Following translocation into the cytoplasm, MHC class I molecules are ubiquitinated and degraded by the proteasome.

[0208] In addition to class I molecules, US2 also leads to the degradation of two proteins of the class II pathway, DR-α and DM-α, as well as HFE, a nonclassical major histocompatibility complex (MHC) class I protein involved in iron regulation.

[0209] The lumenal domain of US2 is responsible for binding to MHC class I and class II molecules, and the transmembrane domain (TM) and cytoplasmic domain (CT) interact with cellular components of the ERAD mechanism or pathway and contribute to translocation and promote the 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), which is an essential component of the US2-dependent MHC I dislocation complex (Loureiro J et al., 2006), which is necessary 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)). Furthermore, US2 interacts with the ER-resident RING-type E3 ligase TRC8 through its TM domain, which also facilitates the ubiquitination and proteasomal degradation of US2-tail-anchored MHC I and II molecules (Stagg HR et al., 2009).

[0210] 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 chains, while the TM domain of US11 binds to Derlin-1. Therefore, 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), which then induces their translocation to the cytosol for proteasomal degradation. Furthermore, US11 activates unfolded proteins. Through Derlin-1, US11 associates with TMEM129, an E3 ligase in the ERAD ring, and recruits Ube2J2 to ubiquitinate MHC-I prior to US11-induced degradation.

[0211] In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US2 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 48, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has the function of endoplasmic reticulum retention. In some embodiments, the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US2 is as shown in SEQ ID NO. 48.

[0212] In some preferred embodiments, the ERAD degradation domain of US2 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence shown in SEQ ID NO. 76, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has the function of endoplasmic reticulum retention. In some embodiments, the amino acid sequence of the ERAD degradation domain of US2 is as shown in SEQ ID NO. 76.

[0213] In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US11 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 54, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US11 is as shown in SEQ ID NO. 54.

[0214] In some preferred embodiments, the ERAD degradation domain of US11 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence shown in SEQ ID NO. 82, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has the function of endoplasmic reticulum retention. In some embodiments, the amino acid sequence of the ERAD degradation domain of US11 is as shown in SEQ ID NO. 82.

[0215] b) HCMV US3

[0216] Rather than promoting degradation of MHC proteins, the HCMV US3 glycoprotein physically associates with peptide-loaded MHC class I heterodimers, resulting in retention of the class I complex in the ER and inhibiting binding of the invariant chain to class II DR-αβ dimers in the ER, leading to mislocalization of the class II complex and reduced peptide loading. Thus, during the early stages of HCMV infection, US3 interferes with the intracellular trafficking and maturation of MHC class I molecules. US3 is an ER-resident membrane protein. Domain swapping experiments have demonstrated that the lumenal domain of US3 is sufficient for US3 itself to remain in the ER, while both the lumenal domain and the transmembrane domain are required for ER retention of MHC class I molecules.

[0217] In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US3 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 51, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US3 is as shown in SEQ ID NO. 51.

[0218] In some preferred embodiments, the ERAD degradation domain of US3 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence shown in SEQ ID NO. 79, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has the function of endoplasmic reticulum retention. In some embodiments, the amino acid sequence of the ERAD degradation domain of US3 is as shown in SEQ ID NO. 79.

[0219] c) HCMV US10

[0220] The HCMV US10 glycoprotein also interacts with components of the MHC class I antigen presentation complex. US10 binds to free class I heavy chains and delays their trafficking from the ER. However, US10 does not affect US2 or US11.

[0221] In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US10 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 57, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US10 is as shown in SEQ ID NO. 57.

[0222] d)HCMV US6, HSV ICP47, CPXV012, EBV BNFL2a, BHV UL49.5

[0223] Unlike US2, US3, US10 and US11, the HCMV L protein US6 affects antigen presentation through a completely different strategy. Instead of 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 causes a conformational change, thereby preventing the binding of ATP. Residues 89-108 in the ER-luminal domain of US6 contribute to the binding of US6 to TAP and are necessary and sufficient for this inhibition. This inhibition of TAP activity not only affects the expression of classical MHC class I alleles, but also affects the expression of non-classical alleles HLA-C and HLA-G in fetal cytotrophoblast cells.

[0224] HSV ICP47, a HCMV US6 protein, is expressed early in the infectious cycle and is dispensable for in vitro replication. Similar strategies can be applied to block class I molecule assembly. ICP47 blocks TAP-mediated peptide transport and binds tightly to the TAP1-TAP2 complex. A clue to the mechanism by which ICP47 blocks TAP is its high species selectivity. Both HSV1 and HSV2 ICP47 inhibit simian, monkey, porcine, dog, and bovine TAP, with little effect on mouse, rat, guinea pig, or rabbit TAP. The affinity of ICP47 for human TAP is approximately 100-fold higher than for mouse TAP. ICP47 inhibits peptide binding to TAP but does not affect ATP binding. With an affinity for TAP 10- to 1000-fold higher than that of most peptides, ICP47 acts as a competitive inhibitor of peptide binding to TAP and is thought to bind directly to the peptide binding site.

[0225] In some preferred embodiments, the TAP binding domain of HHV-7 US6 comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO. 65, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity, and more preferably an amino acid sequence having 98% or 99% or more identity, and has the function of endoplasmic reticulum retention. In some embodiments, the amino acid sequence of the TAP binding domain of HHV-7 US6 is as shown in SEQ ID NO. 65.

[0226] In some preferred embodiments, the TAP binding domain of HSV ICP47 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 67, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the TAP binding domain of HSV ICP47 is as shown in SEQ ID NO. 67.

[0227] In some preferred embodiments, the TAP-binding domain of CPXV012 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 69, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the TAP-binding domain of CPXV012 is as shown in SEQ ID NO. 69.

[0228] In some preferred embodiments, the full-length sequence of CPXV012 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.68, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of CPXV012 is as shown in SEQ ID NO.68.

[0229] In some preferred embodiments, the TAP-binding domain of EBV BNFL2a comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 71, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the TAP-binding domain of EBV BNFL2a is as shown in SEQ ID NO. 71.

[0230] In some preferred embodiments, the full-length sequence of EBV BNFL2a comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.70, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the full-length sequence of EBV BNFL2a is as shown in SEQ ID NO.70.

[0231] In some preferred embodiments, the TAP-binding domain of BHV UL49.5 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 73, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the TAP-binding domain of BHV UL49.5 is as shown in SEQ ID NO. 73.

[0232] In some preferred embodiments, the full-length sequence of the BHV UL49.5 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.72, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the full-length sequence of the BHV UL49.5 is as shown in SEQ ID NO.72.

[0233] e) Others

[0234] In some preferred embodiments, the ERAD degradation domain of HHV-7 US21 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 63, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the ERAD degradation domain of HHV-7 US21 is as shown in SEQ ID NO. 63.

[0235] In some preferred embodiments, the full-length sequence of HHV-7 US21 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.61, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of HHV-7 US21 is as shown in SEQ ID NO.61.

[0236] In some preferred embodiments, the ERAD degradation domain of HCMV UL16 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 85, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the ERAD degradation domain of HCMV UL16 is as shown in SEQ ID NO. 85.

[0237] In some preferred embodiments, the full-length sequence of HCMV UL16 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 83, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of HCMV UL16 is as shown in SEQ ID NO. 83.

[0238] In some preferred embodiments, the ERAD degradation domain of HCMV UL141 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 88, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the ERAD degradation domain of HCMV UL141 is as shown in SEQ ID NO. 88.

[0239] In some preferred embodiments, the full-length sequence of HCMV UL141 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 86, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of HCMV UL141 is as shown in SEQ ID NO. 86.

[0240] In some preferred embodiments, the Golgi retention domain of HCMV UL142 comprises an amino acid sequence that is 80% or greater identical to the amino acid sequence of SEQ ID NO. 91, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identical, and more preferably an amino acid sequence that is 98% or greater identical, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the Golgi retention domain of HCMV UL142 is as shown in SEQ ID NO. 91.

[0241] In some preferred embodiments, the HIV Nef comprises an amino acid sequence that is 80% or greater identical to the amino acid sequence of SEQ ID NO. 92, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identical, and more preferably an amino acid sequence that is 98% or greater identical, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the HIV Nef is as shown in SEQ ID NO. 92.

[0242] In some preferred embodiments, the full-length sequence of HCMV UL142 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 89, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of HCMV UL142 is as shown in SEQ ID NO. 89.

[0243] In some preferred embodiments, the HIV Vpu comprises an amino acid sequence that is 80% or greater identical to the amino acid sequence of SEQ ID NO. 93, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identical, and more preferably an amino acid sequence that is 98% or greater identical, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the HIV Vpu is as shown in SEQ ID NO. 93.

[0244] In some preferred embodiments, the HHV-8 KK3 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence shown in SEQ ID NO. 94, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the HHV-8 KK3 is as shown in SEQ ID NO. 94.

[0245] In some preferred embodiments, the HHV-8 KK5 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence shown in SEQ ID NO. 95, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the HHV-8 KK5 is as shown in SEQ ID NO. 95.

[0246] In some preferred embodiments, the MHV-68 MK3 comprises an amino acid sequence that is 80% or greater identical to the amino acid sequence of SEQ ID NO. 96, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identical, and more preferably an amino acid sequence that is 98% or greater identical, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the MHV-68 MK3 is as shown in SEQ ID NO. 96.

[0247] In some preferred embodiments, the HTLV-1 p12 comprises an amino acid sequence that is 80% or greater identical to the amino acid sequence of SEQ ID NO. 97, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identical, and more preferably an amino acid sequence that is 98% or greater identical, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the HTLV-1 p12 is as set forth in SEQ ID NO. 97.

[0248] In some preferred embodiments, the KDEL receptor-binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 100, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the KDEL receptor-binding domain of the vaccinia virus protein CPXV203 is as shown in SEQ ID NO. 100.

[0249] In some preferred embodiments, the KDEL receptor-binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 103, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the KDEL receptor-binding domain of the vaccinia virus protein CPXV203 is as shown in SEQ ID NO. 103.

[0250] In some preferred embodiments, the full-length sequence of the vaccinia virus protein CPXV203 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 98, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the full-length sequence of the vaccinia virus protein CPXV203 is as shown in SEQ ID NO. 98.

[0251] In some preferred embodiments, the full-length sequence of the vaccinia virus protein CPXV203 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.101, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the full-length sequence of the vaccinia virus protein CPXV203 is as shown in SEQ ID NO.101.

[0252] 2. Targeting domain

[0253] The target binding domain of the chimeric protein constructs provided herein can be any structure that recognizes and binds to a target protein, such as an antibody, an antibody fragment, a functional motif derived from a natural protein (e.g., a natural ligand), an artificially synthesized polypeptide or protein that has affinity for the target protein, or various variants of the aforementioned molecules, such as mutants, fusions, and truncations. For example, the targeting domain can be a natural ligand of the target protein, an antibody or antigen-binding fragment thereof that specifically recognizes the target protein, or an antigen that can be specifically recognized by an antibody.

[0254] In some embodiments, the targeting domain comprises an antibody or a functional fragment thereof that specifically targets a target protein (e.g., a diabody, Fab, Fab', F(ab')2, Fd, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (dsdiabody), a single-chain antibody molecule (scFv), a scFv dimer (bivalent diabody), Fv (scFv), a multispecific antibody (e.g., a bispecific antibody), a camelized single domain antibody (e.g., VHH), a nanobody, a domain antibody, a bivalent domain antibody, a diabody, a triabody, and a tetrabody). The targeting protein domain may also comprise any binding ligand capable of binding to a target protein, including but not limited to a ligand, a portion of a receptor that binds to a ligand, a portion of a viral protein that binds to a host protein, and the like.

[0255] The target protein can be any protein whose level or activity is desired to be modulated, for example, by altering its activity through the ERAD mechanism to block proteins in the ER and accelerate proteasome-mediated degradation, the degradation of which can change the phenotype of the cell, improve cell function, induce cell apoptosis and / or inhibit viral replication, etc., thereby achieving therapeutic purposes (e.g., improving efficacy, reducing side effects, etc.). In some embodiments, the target protein can be a protein involved in the cell cycle, apoptosis, signal transduction, cell differentiation, cell dedifferentiation, cell growth, the production of cytokines or their biological regulators, the production of cytokines or biological regulators, the production of regulatory or functional proteins, pro-inflammatory signaling, or glucose regulation pathways.

[0256] In another embodiment, the target protein is a disease-associated protein (also referred to as a "pathogenic protein"). The "disease-associated protein" or "pathogenic protein" described herein refers to any protein whose function or activity changes will lead to the occurrence of the disease, or whose function is important for the development of the disease. Exemplary pathogenic proteins include but are not limited to oncoproteins, viral proteins, and autoimmune disease-causing proteins (e.g., antibodies produced by plasma cells).

[0257] In some embodiments, the target protein is selected from: target proteins related to immune function, target proteins related to nervous system diseases, target proteins related to infectious diseases (such as target proteins related to viral infections), target proteins related to self-antigens or target proteins related to tumors (or oncogenic proteins), and target proteins related to metabolic diseases.

[0258] In some embodiments, the pathogenic protein is an oncoprotein. The oncoprotein may be encoded by an oncogene, including but not limited to 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, CDKN 1A, 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 / F LI1, FES, FGF3, FER, FGR, FLI1 / ERGB2, FOS, FPS / FES, FRA1, FRA2, FYN, HCK, HEK, HER3 / ERBB-2, ERBB-3, HER4 / ERBB-4, HST2, I NK4A, INK4B, JUN, JUNB, JUND, KIP2, KIT, KRAS2A, KRAS2B, LCK, LYN, MAS, MAX, MCC, MLH1, MOS, MSH2, MYBA, MYBB, NF1, NF2, P5 3. 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, EL K3, 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,In some embodiments, the oncoprotein may be a member of the Bcl-2 family (e.g., Bcl-2, Bcl-xL, and Bcl-xL). Bcl-w), VEGF / VEGFR, PDGFRβ, EGFR, EGFR mutant, IGF-1R, HDACs, HER2, MYC, KRAS, AFP, CEA, CA199, estrogen receptor ER-α, androgen receptor 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.

[0259] In some embodiments, the target protein includes immune function-related proteins. As used herein, the term "immune function-related proteins" refers to functional proteins 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.), immunostimulatory / co-stimulatory molecules (e.g., CD3, CD80 / 86, CD28, etc.), and other molecules involved in innate or adaptive immunity. In some embodiments, target proteins associated with immune function include, for example, CD123, CD7, CD5, MHC class I molecules, MHC class II molecules, non-classical MHC molecules (such as 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 indoleamine-2,3-dioxygenase (IDO), or arginase 1 (ARG1).

[0260] In some embodiments, the target protein includes a target protein associated with a nervous system disease. As used herein, the term "nervous system disease associated" refers to a protein involved in a nervous system disease, particularly a protein in a neurodegenerative disease. In some embodiments, the target protein associated with a nervous system disease 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).

[0261] In some embodiments, the target protein includes a target protein associated with viral infection. The target protein associated with viral infection includes, for example, 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, RNA-dependent RNA polymerase (RdRp), such as SARS-CoV-2 RNA-dependent RNA polymerase, herpesvirus DNA and RNA polymerase, herpesvirus capsid glycoprotein, CMV DNA polymerase, CMV capsid glycoprotein, RSV surface protein, RSV capsid protein, RSV RNA polymerase, influenza virus RNA polymerase, influenza virus envelope protein, HPV DNA polymerase, HPV capsid protein. In another embodiment, the target protein is a viral protein, such as HBV surface antigen, HBeAg, HBV polymerase protein, HIV Gag protein, HIV Env protein, etc., whose function is believed to be important for viral replication, amplification and function and the progression of viral diseases.

[0262] In some embodiments, the target protein comprises a target protein associated with an autoantigen.A variety of autoantigens associated with autoimmune diseases are known in the art. For example, autoantigens associated with type 1 diabetes mellitus, such as islet cell antigen (ICA), insulin (IAA), glutamic acid decarboxylase 65 (GAD65), and insulinoma antigen-2 (IA-2); autoantigens associated with rheumatoid arthritis (RA), such as citrullinated protein / peptide antibodies, heterogeneous nuclear ribonucleoprotein A2 / B1, aldolase, alpha-enolase, calreticulin, heat-activated protein (HSP60), BiP, PGK1, stress-induced phosphoprotein 1, and FUSE-BP1 / 2; autoantigens associated with systemic lupus erythematosus (SLE), such as deoxyribonucleoprotein, SmD1 and SmD3, Clq, sore anticoagulant (LA), cardiolipin (CL), β2-glycoprotein I (β2GP I), prothrombin (PT), and phosphatidylserine (PS); and autoantigens associated with systemic sclerosis (SLE). Sclerosis (SSc) / scleroderma (SD)-related autoantigens, such as Scl-70, SSA, Ro52; antigens associated with autoimmune liver disease, such as mitochondrial antigens, Sp100, PML, gp210, p62; autoantigens associated with myasthenia gravis, such as acetylcholine receptor; autoantigens associated with central nervous system autoimmune diseases (limbic encephalitis, encephalomyelitis, cerebellar ataxia), such as voltage-gated potassium channel (VGKC) complex, voltage-gated calcium channel receptor, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor, γ-aminobutyric acid-B (GABAB) receptor, glycine receptor; autoantigens associated with multiple sclerosis, such as Myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG); autoantigens associated with polymyositis (PM) and dermatomyositis (DM), such as Jo-1, Mi-2, PM-Scl, and Ro-52; autoantigens associated with gluten-sensitive enteropathy, such as endomysial antibodies (EMA) and tissue transglutaminase (tTG); autoantigens associated with anti-NMDAR antibody encephalitis, such as N-methyl-D-aspartate receptor; autoantigens associated with neuromyelitis optica (NMO), such as aquaporin 4 (AQP4); autoantigens related to reproduction, such as ovarian antigens and sperm antigens.

[0263] In some embodiments, the pathogenic protein is a target protein associated with a metabolic disease. The target proteins related to metabolic diseases include, for example, target proteins for atherosclerosis (AS), including but not limited to CD36, low-density lipoprotein receptor (LDLR), ChemR23 (CMKLR1), and mitochondrial dehydrogenase ALDH4A1 (target protein); target proteins for type 2 diabetes, including but not limited to RalGAPα1 and dipeptidyl peptidase IV (DPP4); target proteins for non-alcoholic fatty liver disease, including but not limited to TMEM16A and VAMP3; target proteins for tumor glucose metabolism, including but not limited to hexokinase (HK), glucose transporter 1 (GLUT1), glucose transporter 4 (GLUT4), phosphoglycerate dehydrogenase, and lactate dehydrogenase; target proteins for tumor lipid metabolism, including but not limited to ATP citrate lyase (ACLY), fatty acid synthase (FAS), and ATP citrate lyase (ACLY). synthase, FASN), cholesterol esterase (acetyl-CoA acetyltransferase 1, ACAT1).

[0264] In some embodiments, the targeting domain specifically targets a target protein other than the following target proteins: TCR, HLA-I, MICA, and MICB, and the viral endoplasmic reticulum-resident protein is any viral endoplasmic reticulum-resident protein described in the present disclosure.

[0265] In some embodiments, the targeting domain specifically targets at least two of the following target proteins: TCR, HLA-I, MICA, MICB, and the viral endoplasmic reticulum-resident protein is any viral endoplasmic reticulum-resident protein described in the present disclosure.

[0266] In some embodiments, the targeting domain specifically targets TCR, HLA-I, MICA, or MICB, and the viral endoplasmic reticulum-resident protein is not adenoviral E3-K19.

[0267] In some embodiments, the targeting domain of the chimeric protein construct is connected to the ERAD machinery protein binding domain via a hinge or linker. In some embodiments, the hinge comprises the amino acid sequence shown in SEQ ID NO. 123 (IgG4 hinge) or SEQ ID NO. 133 ((Gly4Ser)2).

[0268] In some preferred embodiments, the HLA binding domain of the HCMV glycoprotein US2 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.47, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the HLA binding domain of the HCMV glycoprotein US2 is as shown in SEQ ID NO.47.

[0269] In some preferred embodiments, the HLA binding domain of US2 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.75, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the HLA binding domain of US2 is as shown in SEQ ID NO.75.

[0270] In some preferred embodiments, the MHC binding domain of the HCMV glycoprotein US11 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.53, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the MHC binding domain of the HCMV glycoprotein US11 is as shown in SEQ ID NO.53.

[0271] In some preferred embodiments, the HLA binding domain of US11 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.81, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the HLA binding domain of US11 is as shown in SEQ ID NO.81.

[0272] In some preferred embodiments, the HLA binding domain of the HCMV glycoprotein US3 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.50, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the HLA binding domain of the HCMV glycoprotein US3 is as shown in SEQ ID NO.50.

[0273] In some preferred embodiments, the HLA binding domain of US3 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.78, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the HLA binding domain of US3 is as shown in SEQ ID NO.78.

[0274] In some preferred embodiments, the HLA binding domain of the HCMV glycoprotein US10 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.56, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the HLA binding domain of the HCMV glycoprotein US10 is as shown in SEQ ID NO.56.

[0275] In some preferred embodiments, the MHC I and / or MHC II binding protein molecule domain or its functional variant 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.

[0276] In some preferred embodiments, the binding protein molecular domain targeting MHC I and / or MHC II or its functional variant further comprises a viral protein that directs the inhibition or degradation of the NK target protein of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 or ULBP6; preferably, the viral protein is selected from HCMV UL16, UL141, UL142 or adenovirus E3-19K.

[0277] In some preferred embodiments, the MHC I and / or MHC II binding protein molecular domain or its functional variant further comprises a viral protein that transports MHC I molecules from the Golgi apparatus to the lysosome 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.

[0278] In some preferred embodiments, the MHC I and / or MHC II-targeting binding protein molecular domain or its functional variant further comprises a viral protein that mediates the return of MHC-polypeptide molecules from the Golgi apparatus to the endoplasmic reticulum and promotes their 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.

[0279] In some preferred embodiments, the MHC binding domain of HHV-7 US21 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 62, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the MHC binding domain of HHV-7 US21 is as shown in SEQ ID NO. 62.

[0280] In some preferred embodiments, the NK target protein binding domain of HCMV UL16 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown 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 99% or more identity; the amino acid sequence of the NK target protein binding domain of HCMV UL16 is as shown in SEQ ID NO.84.

[0281] In some preferred embodiments, the NK target protein binding domain of HCMV UL141 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown 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 99% or more identity; the amino acid sequence of the NK target protein binding domain of HCMV UL141 is as shown in SEQ ID NO.87.

[0282] In some preferred embodiments, the MICA and ULBP3 binding domain of HCMV UL142 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown 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 99% or more identity; the amino acid sequence of the MICA and ULBP3 binding domain of HCMV UL142 is as shown in SEQ ID NO. 90.

[0283] In some preferred embodiments, the MHC binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 99, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the MHC binding domain of the vaccinia virus protein CPXV203 is as shown in SEQ ID NO. 99.

[0284] In some preferred embodiments, the MHC binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO. 102, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the MHC binding domain of the vaccinia virus protein CPXV203 is as shown in SEQ ID NO. 102.

[0285] In some preferred embodiments, the MHC binding domain of the adenovirus E3-19K comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 59, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the MHC binding domain of the adenovirus E3-19K is as shown in SEQ ID NO. 59.

[0286] 3. Protein degradation pathway member binding domain

[0287] The chimeric protein constructs provided by the present disclosure may also comprise a protein degradation pathway member binding domain. As used herein, a "protein degradation pathway member binding domain" refers to any portion that is capable of directly or indirectly binding to a member of a protein degradation pathway.

[0288] The protein degradation pathway can be any pathway that can mediate protein degradation in cells. Known protein degradation pathways include, but are not limited to, ubiquitination-proteasome pathways, endosome-lysosome pathways, and autophagy degradation pathways.

[0289] Members of the ubiquitination-proteasome pathway include, for example, E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, E3 ubiquitin ligases, and the proteasome.

[0290] Examples of E1 ubiquitin-activating enzymes include, e.g., UBA1, UBA2, UBA3, UBA5, UBA6, UBA7, ATG7, NAE1, and SAE1.

[0291] Examples of E2 ubiquitin-conjugating enzymes include, e.g., 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.

[0292] 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, beta TRCP1, HUWEI, TRAF6, SMURF1, and E6AP. In certain 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, TOPO RS, TRIP12, UBE3A, UBE3B, UBE3C, UBE3D, UBE4A, UBE4B, UBOX5, UBR5, VHL, WWP1, WWP2, Parkin, and MKRN1.

[0293] Members of the endosomal-lysosomal pathway include, for example, AP-1, AP-2, AP-3, endosomes, lysosomes, HOPS, ESCRT, GASP, BLOC-1, ESCRT, Retromer, ESCRT, sortingnexin, Dapper2, SNX4, Pincher, Rap1-PDZ-GEF1, clathrin, C3G / CrkL / Shp2 / Gab2, etc.

[0294] Members of the autophagy degradation pathway include, for example, 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, NUFIP1, etc.

[0295] In some embodiments, the protein degradation pathway member binding domain is a VHL binding domain. In some embodiments, the VHL binding domain comprises an amino acid sequence such as SEQ ID NO. 119 (DRHDSGLDSM) or SEQ ID NO. 120 (ALAPYIP).

[0296] 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).

[0297] In some embodiments, the protein degradation pathway member binding domain is an E3 ubiquitin ligase binding domain comprising an amino acid sequence as shown in SEQ ID NO. 119 (DRHDSGLDSM).

[0298] 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).

[0299] 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 group consisting of: yeast Rad23 (e.g., S. cerevisiae Rad23), the ubiquitin-like (UbL) domain of human Rad23b (hHR23b), HPV E7, and the proteasome binding domain of 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.

[0300] The inventors of the present application have found that compared with the basic TPD design (Targeted protein binding domain-Transmembrane domain-ER retention domain, TBD-TMD-ERD), 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)) (for example, those described above) have significantly improved degradation effects on target proteins. The functional structure of viral ER-resident proteins has different binding abilities to the ERAD reverse transporter complex, and the natural structure also interacts differently with the ER-resident E3 ligase. At the same time, it also has to compete with the normal ERAD degradation pathway of misfolded proteins in the endoplasmic reticulum. Therefore, the natural TMD-ERD domain has differences and limitations in its ability to ubiquitinate and degrade target proteins. In order 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 target proteins transported to the cytoplasm, or transport them to lysosomes to achieve efficient hydrolysis of target proteins.

[0301] In some embodiments, the targeting domain or ERAD machinery protein binding domain of the chimeric protein construct is connected to the protein degradation pathway member binding domain via a hinge or linker. The hinge or linker can be one or more molecular sequence structures (Glyx ​​Ser) n, where n can be a number from 1 to 10, and x, y can be a number from 0 to 10, respectively, but x, y cannot be 0 at the same time. In some embodiments, the hinge comprises an amino acid sequence as shown in SEQ ID NO. 172 (IgG4 hinge) or SEQ ID NO. 173 ((Gly4Ser) 2).

[0302] 4. TPD chimeric protein construct

[0303] In some embodiments, the present disclosure provides a chimeric protein construct comprising a TCR-targeting protein binding domain and adenovirus E3-19K and one or more protein degradation pathway member binding domains described above. In some embodiments, the present disclosure provides a chimeric protein construct comprising a TCR-targeting protein binding domain and adenovirus E3-19K and an E3 ubiquitin ligase binding domain. In some embodiments, the E3 ubiquitin ligase binding domain comprises the amino acid sequence shown in SEQ ID NO. 147 (DRHDSGLDSMGSGSGALAPYIP).

[0304] In some embodiments, the chimeric protein construct provided by the present disclosure comprises a targeting protein binding domain that targets TCR and a transmembrane domain or a functional variant thereof and an endoplasmic reticulum retention domain of a viral endoplasmic reticulum resident protein other than adenovirus E3-19K. The viral endoplasmic reticulum resident protein can be at least one selected from the group consisting of HCMV glycoprotein 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 vaccinia virus protein CPXV203.

[0305] In some embodiments, the chimeric protein construct provided by the present disclosure comprises a targeting protein binding domain that targets the above-mentioned target protein (e.g., TCR) and a transmembrane domain or a functional variant thereof and an endoplasmic reticulum retention domain or a functional variant thereof of HCMV US2 and / or US11.

[0306] In some embodiments, the chimeric protein construct provided by the present disclosure comprises a targeting protein binding domain that targets the above-mentioned target protein (e.g., TCR) and the transmembrane domain or a functional variant thereof and the endoplasmic reticulum retention domain or a functional variant thereof of HCMV US3.

[0307] In some embodiments, the chimeric protein construct provided by the present disclosure comprises a targeting protein binding domain that targets the above-mentioned target protein (e.g., TCR) and the transmembrane domain or a functional variant thereof and the endoplasmic reticulum retention domain or a functional variant thereof of HCMV US10.

[0308] In some embodiments, the chimeric protein constructs provided by the present disclosure comprise a targeting protein binding domain that targets the above-mentioned target protein (e.g., TCR) and a transmembrane domain or a functional variant thereof of HCMV US6, HSV ICP47, CPXV012, EBV BNFL2a and / or BHV UL49.5 and an endoplasmic reticulum retention domain or a functional variant thereof.

[0309] In some embodiments, the chimeric protein construct provided by the present disclosure comprises a targeting protein binding domain that targets the above-mentioned target protein (e.g., TCR) and the transmembrane domain or a functional variant thereof and the endoplasmic reticulum retention domain or a functional variant thereof of HHV-7 US21.

[0310] In some embodiments, the chimeric protein constructs provided by the present disclosure comprise a targeting protein binding domain that targets the above-mentioned target protein (e.g., TCR) and a transmembrane domain or a functional variant thereof and an endoplasmic reticulum retention domain or a functional variant thereof of HCMV UL16, UL141 and / or UL142.

[0311] In some embodiments, the chimeric protein constructs provided by the present disclosure comprise a targeting protein binding domain that targets the above-mentioned target protein (e.g., TCR) and a transmembrane domain or a functional variant thereof and an endoplasmic reticulum retention domain or a 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.

[0312] In some embodiments, the chimeric protein construct provided by the present disclosure comprises a targeting protein binding domain that targets the above-mentioned target protein (e.g., TCR) and a transmembrane domain or a functional variant thereof and an endoplasmic reticulum retention domain or a functional variant thereof of the Cowpox Virus protein CPXV203.

[0313] In some embodiments, the chimeric protein construct provided by the present disclosure includes a targeting protein binding domain and adenovirus E3-19K that is not targeted to TCR. In some embodiments, the chimeric protein construct provided by the present disclosure includes a targeting protein binding domain and adenovirus E3-19K that targets CD123.

[0314] In some embodiments, the chimeric protein construct further comprises one or more protein degradation pathway member binding domains as described above.

[0315] 5. Co-expression

[0316] In some embodiments, the chimeric protein constructs provided herein and at least one co-expression moiety are co-expressed. Any suitable method for co-expression may be used. For example, the chimeric protein construct (or a portion thereof) and the at least one co-expression moiety may be connected by a cleavable linker, such that upon cleavage of the linker, the chimeric protein construct and the co-expression moiety can be co-expressed.

[0317] In some embodiments, the chimeric protein constructs provided herein and at least one co-expression moiety are linked, for example, by a self-cleavable linker. In some embodiments, the self-cleavable linker is a cleavable peptide; for example, a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, a F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide.

[0318] In some preferred embodiments, the T2A comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.108, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of T2A is as shown in SEQ ID NO.108.

[0319] In some preferred embodiments, the amino acid sequence of the GSG-T2A peptide is shown as SEQ ID NO.109.

[0320] In some preferred embodiments, the P2A comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.110, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the P2A is as shown in SEQ ID NO.110.

[0321] In some preferred embodiments, the amino acid sequence of the GSG-P2A peptide is shown as SEQ ID NO.111.

[0322] In some preferred embodiments, the E2A comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.112, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the E2A is as shown in SEQ ID NO.112.

[0323] In some preferred embodiments, the amino acid sequence of the GSG-E2A peptide is shown as SEQ ID NO.113.

[0324] In some preferred embodiments, the F2A comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown 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 99% or more identity; the amino acid sequence of F2A is shown in SEQ ID NO. 114.

[0325] In some preferred embodiments, the amino acid sequence of the GSG-F2A peptide is shown as SEQ ID NO.115.

[0326] The coexpression portion provided herein can be any protein or polypeptide with a biological function. Depending on the biological function desired to be achieved, a suitable coexpression portion can be selected. For example, in order to reduce the immunogenicity or antigen presentation of the cell, a protein that can degrade or reduce MHC class I or class II molecules can be selected as a coexpression portion. For another example, in order to enable the cell to recognize the target protein, a binding domain (such as a chemokine receptor or a chimeric antigen receptor CAR) that recognizes or binds to the target protein can be selected as a coexpression portion. For another example, in order to increase immunostimulatory activity, an immunostimulatory molecule can be selected as a coexpression portion.

[0327] In some preferred embodiments, examples of the co-expressed moiety include, but are not limited to, complete viral ER resident glycoproteins (e.g., HCMV US2, US3, US11, US10, adenovirus E3-K19, 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 cytotoxicity receptors, TRAIL, DNAM-1, CD16a, 2B4, NTB-A, CRACC, and NKp80), CNK signaling transduction components, cytokines, CD7, immunostimulatory molecules (e.g., TNF-α, IFN-β, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL- 18 and one or more of granulocyte macrophage colony stimulating factor), etc.

[0328] i) Intact viral ER-resident glycoprotein

[0329] In some embodiments, the co-expressed moiety is an intact viral ER-resident glycoprotein, including but not limited to, HCMV US2, US3, US11, US10, adenovirus E3-K19, HCMV US6, and HSV ICP47.

[0330] In some preferred embodiments, the full-length sequence of the HCMV glycoprotein US2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown 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 99% or more identity, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US2 is as shown in SEQ ID NO.46.

[0331] In some preferred embodiments, the full-length sequence of US2 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.74, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of US2 is as shown in SEQ ID NO.74.

[0332] In some preferred embodiments, the full-length sequence of the HCMV glycoprotein US3 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.49, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US3 is as shown in SEQ ID NO.49.

[0333] In some preferred embodiments, the full-length sequence of US3 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.77, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of US3 is as shown in SEQ ID NO.77.

[0334] In some preferred embodiments, the full-length sequence of the HCMV glycoprotein US11 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.52, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US11 is as shown in SEQ ID NO.52.

[0335] In some preferred embodiments, the full-length sequence of US11 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.80, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of US11 is as shown in SEQ ID NO.80.

[0336] In some preferred embodiments, the full-length sequence of the HCMV glycoprotein US10 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown 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 99% or more identity, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US10 is as shown in SEQ ID NO.55.

[0337] In some preferred embodiments, the full-length sequence of the adenovirus E3-19K comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown 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 99% or more identity, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of the adenovirus E3-19K is as shown in SEQ ID NO.58.

[0338] In some preferred embodiments, the full-length sequence of HCMV US6 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown 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 99% or more identity, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of HCMV US6 is as shown in SEQ ID NO.64.

[0339] In some preferred embodiments, the full-length sequence of HSV ICP47 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.66, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of HSV ICP47 is as shown in SEQ ID NO.66.

[0340] ii) Chimeric Antigen Receptor (CAR)

[0341] In certain embodiments, the co-expression portion is a chimeric antigen receptor (CAR). The CAR comprises a target protein binding domain (e.g., an extracellular recognition domain targeting a tumor), a transmembrane domain, and an immunoreceptor activation signaling domain (ITAM) (also referred to as an "intracellular signaling domain"). In certain embodiments, the CAR may further comprise a costimulatory domain. In certain embodiments, a hinge or joint is included between the extracellular recognition domain, the transmembrane domain, and / or the intracellular signaling domain targeting a tumor.

[0342] In some embodiments, the tumor-targeting extracellular recognition domain is selected from a tumor antigen-specific binding domain, a tumor microenvironment target antigen binding domain, and / or a tumor microenvironment-targeting chemotactic receptor.

[0343] In some preferred embodiments, the tumor-targeting extracellular recognition domain is selected from an antibody or a functional fragment thereof, a TCR or a combination thereof that can target and recognize tumor-associated antigens; the functional fragment of the antibody is selected from Fd, Fv, Fab, Fab', F(ab')2, Fv (scFv), a single-chain antibody (scFv) or a nanobody, a double-chain antibody, a three-chain antibody and a four-chain antibody.

[0344] In some preferred embodiments, the transmembrane domain of the CAR described herein can be derived from any membrane-bound protein or transmembrane protein, including but not limited to BAFFR, BLAME (SLAMF8), CD2, CD3ε, CD4, CD5, CD8, CD9, CD11a (CD18, ITGAL, LFA-l), CD11b, CD11c, CD11d, CD16, CD19, CD22, CD27, CD28, CD29, CD33, CD37, CD40, CD45, CD49a, CD49d, CD49f, CD64, CD80, CD84, CD86, CD96(Tactile), CD100(SEMA4D), CD103, CD134, CD137(4-1BB), CD150(IPO-3, SLAMF1, SLAM), CD154, CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(Ly9), CD244(2B4, SLAMF4), CD278(ICOS), CEACAM1, CRT AM, GITR, HYEM (LIGHTR), IA4, IL2Rβ, IL2Rγ, IL7R α, 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, α, β, or ζ chain of the T cell receptor, TNFR2, VLA1, and VLA-6.

[0345] In some preferred embodiments, the transmembrane domain of the CAR is selected from the group consisting of an NK cell activating receptor transmembrane domain, 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, and a BTLA transmembrane domain, and combinations thereof.

[0346] In some preferred embodiments, the transmembrane domain of the CAR is selected from the group consisting of a CD8 transmembrane domain, an α and / or β chain transmembrane domain of a T cell receptor, a CD28 transmembrane domain, a CD3ε transmembrane domain, a CD45 transmembrane domain, a CD4 transmembrane domain, a CD5 transmembrane domain, a CD8 transmembrane domain, a CD9 transmembrane domain, a CD16 transmembrane domain, a CD22 transmembrane domain, a CD33 transmembrane domain, a CD37 transmembrane domain, a CD64 transmembrane domain, a CD80 transmembrane domain, a CD86 transmembrane domain, a CD134 transmembrane domain, a CD137 transmembrane domain, a CD154 transmembrane domain, a GITR transmembrane domain, and combinations thereof.

[0347] In some preferred embodiments, the immunoreceptor activation signaling domain (ITAM) is derived from the intracellular activation signaling domain of an immunoreceptor; preferably, the immunoreceptor 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 immunoreceptor activation signaling domain (ITAM) is fused with an NK cell signal transducer or a functional variant thereof; preferably, the immunoreceptor is CD3ζ.

[0348] In some preferred embodiments, the immunoreceptor activation signaling domain (ITAM) is derived from intracellular signaling domains such as CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI CD66d, DAP10 and DAP12.

[0349] In some preferred embodiments, the intracellular signaling domain of the CAR includes the intracellular signaling domain and / or the co-stimulatory signaling domain of an NK cell activating receptor.

[0350] In some preferred embodiments, the T cell co-stimulatory signaling domain is derived from the intracellular signaling domain of a co-stimulatory molecule; preferably, the co-stimulatory molecule is selected from MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, 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, BAFF R, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, 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, CD11c , ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, NCR, DAP10, DAP12, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2 B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM( SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, 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, PTCH2.More preferably, the costimulatory signaling domain can 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.

[0351] In some embodiments, the hinge and / or transmembrane domain of CAR described herein provide the cell surface presentation of the extracellular domain of CAR. The hinge of CAR described herein can be derived from any membrane-bound protein or transmembrane protein, including but not limited to 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, CD49f, CD6 4. CD80, CD84, CD86, CD96 (Tactile), CD100 (SEMA4D), CD103, CD134, CD137 (4-1BB), CD150 (IPO-3, SLAMF1, SLAM), C D154, CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(Ly9), CD244(2B4, SLAMF4), CD278(ICOS), CEACAM1, CRT AM, GITR, HYEM (LIGHTR), IA4, IL2R β, IL2R γ, 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, α, β or ζ chain of T cell receptor, TNFR2, VLA1 and VLA-6. In some embodiments, the hinge of CAR described herein includes the hinge region of CD8 α, the hinge region of human immunoglobulin (Ig) or a glycine-serine-rich sequence.

[0352] In some preferred embodiments, the linker is a flexible linker; preferably, the flexible linker comprises the amino acid sequence shown (Gly(x)Ser(y))n, wherein 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 shown in SEQ ID NO.104 or the amino acid sequence shown in SEQ ID NO.105.

[0353] In some preferred embodiments, the hinge is an IgG1 hinge or an IgG4 hinge.

[0354] In some preferred embodiments, the IgG1 hinge comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.106, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the IgG1 hinge is as shown in SEQ ID NO.106.

[0355] In some preferred embodiments, the IgG4 hinge comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.107, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the IgG4 hinge is as shown in SEQ ID NO.107.

[0356] In some preferred embodiments, a cleavable peptide is contained between the NK activating receptor component, CNK signaling transduction component and / or UT component; for example, a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, a F2A peptide, a GSG-F2A peptide, a P2A peptide or a GSG-P2A peptide.

[0357] In some preferred embodiments, the T2A comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.108, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of T2A is as shown in SEQ ID NO.108.

[0358] In some preferred embodiments, the amino acid sequence of the GSG-T2A peptide is shown as SEQ ID NO.109.

[0359] In some preferred embodiments, the P2A comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.110, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the P2A is as shown in SEQ ID NO.110.

[0360] In some preferred embodiments, the amino acid sequence of the GSG-P2A peptide is shown as SEQ ID NO.111.

[0361] In some preferred embodiments, the E2A comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.112, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the E2A is as shown in SEQ ID NO.112.

[0362] In some preferred embodiments, the amino acid sequence of the GSG-E2A peptide is shown as SEQ ID NO.113.

[0363] In some preferred embodiments, the F2A comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown 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 99% or more identity; the amino acid sequence of F2A is shown in SEQ ID NO. 114.

[0364] In some preferred embodiments, the amino acid sequence of the GSG-F2A peptide is shown as SEQ ID NO.115.

[0365] iii) CNK receptors

[0366] In certain embodiments, the co-expressed portion is a CNK receptor. The CNK receptor comprises a chimeric NK activating receptor component and a chimeric NK signal transduction component (also referred to as a "CNK signal transduction component"). The chimeric NK activating receptor component may comprise at least an NK cell activating receptor or a functional variant thereof.

[0367] NK cell activating receptor

[0368] In some embodiments, the NK cell activating receptor comprises: (a) an NK cell activating receptor extracellular domain (ED) or a functional variant thereof, (b) an NK cell activating receptor transmembrane domain (TMD) or a functional variant thereof, and (c) an NK cell activating receptor intracellular domain (ICD) or a functional variant thereof; Optionally, a hinge or joint is included between the NK cell activating receptor extracellular domain or a functional variant thereof, the NK cell activating receptor transmembrane domain or a functional variant thereof and / or the NK cell activating receptor intracellular domain or a functional variant thereof. A detailed description of NK cell activating receptors and CNK signaling switching components can be found in, for example, US 2020 / 0308248A1, the entire contents of which are incorporated herein by reference.

[0369] 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 attachment 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.

[0370] In some preferred embodiments, the NK cell activating receptor is a NK cell activating receptor of mammalian origin; preferably, the mammal is selected from humans, primates, mice, horses, cows, sheep, goats, cats, pigs, dogs, llamas, alpacas, elephants, squirrels, and guinea pigs.

[0371] In some preferred embodiments, the NK cell activating receptor is a recombinant NK cell activating receptor comprising NK cell activating receptor domains from different sources.

[0372] In some preferred embodiments, the NK cell activating receptor is a human NK cell activating receptor; preferably, the NK cell activating receptor is a recombinant NK cell activating receptor comprising different human NK cell activating receptor domains.

[0373] In some preferred embodiments, the NK cell activating receptor is a murine NK cell activating receptor; preferably, the NK cell activating receptor is a recombinant NK cell activating receptor comprising different murine NK cell activating receptor domains.

[0374] In some preferred embodiments, the NK cell activating receptor is a recombinant NK cell activating receptor comprising human and murine NK cell activating receptor domains.

[0375] 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.

[0376] 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.

[0377] 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.

[0378] 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.

[0379] In some preferred embodiments, the functional variant 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 types.

[0380] In some preferred embodiments, the extracellular domain of human NKG2D comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.1, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the extracellular domain of human NKG2D is as shown in SEQ ID NO.1.

[0381] In some preferred embodiments, the full-length sequence of human NKG2D comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.2, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKG2D is as shown in SEQ ID NO.2.

[0382] In some preferred embodiments, the extracellular domain of mouse NKG2D comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.3, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the extracellular domain of mouse NKG2D is as shown in SEQ ID NO.3.

[0383] In some preferred embodiments, the full-length sequence of mouse NKG2D comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.4, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of mouse NKG2D is as shown in SEQ ID NO.4.

[0384] In some preferred embodiments, the full-length sequence of human-mouse recombinant NKG2D comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.5, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human-mouse recombinant NKG2D is as shown in SEQ ID NO.5.

[0385] In some preferred embodiments, the full-length sequence of human NKG2C comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.6, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKG2C is as shown in SEQ ID NO.6.

[0386] In some preferred embodiments, the full-length sequence of human NKG2E comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 7, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKG2E is as shown in SEQ ID NO.7.

[0387] In some preferred embodiments, the full-length sequence of human NKG2F comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 8, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKG2F is as shown in SEQ ID NO.8.

[0388] In some preferred embodiments, the full-length sequence of human CD94 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence shown in SEQ ID NO. 9, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity; the amino acid sequence is shown in SEQ ID NO. 9.

[0389] In some preferred embodiments, the full-length sequence of human KIR2DL4 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 10, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human KIR2DL4 is as shown in SEQ ID NO.10.

[0390] In some preferred embodiments, the full-length sequence of human KIR2DS1 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 11, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human KIR2DS1 is as shown in SEQ ID NO.11.

[0391] In some preferred embodiments, the full-length sequence of human KIR2DS2 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 12, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human KIR2DS2 is as shown in SEQ ID NO.12.

[0392] In some preferred embodiments, the full-length sequence of human KIR2DS4 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 13, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human KIR2DS4 is as shown in SEQ ID NO.13.

[0393] In some preferred embodiments, the full-length sequence of human KIR3DS1 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 14, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human KIR3DS1 is as shown in SEQ ID NO.14.

[0394] In some preferred embodiments, the full-length sequence of human NKp46 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.15, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKp46 is as shown in SEQ ID NO.15.

[0395] In some preferred embodiments, the full-length sequence of human NKp44 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.16, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKp44 is as shown in SEQ ID NO.16.

[0396] In some preferred embodiments, the full-length sequence of human NKp30 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.17, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKp30 is as shown in SEQ ID NO.17.

[0397] In some preferred embodiments, the full-length sequence of human DNAM1 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 18, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human DNAM1 is as shown in SEQ ID NO.18.

[0398] In some preferred embodiments, the full-length sequence of human TRAIL comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 19, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human TRAIL is as shown in SEQ ID NO.19.

[0399] In some preferred embodiments, the full-length sequence of human CD16a comprises an amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO: 20, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length human CD16a is as shown in SEQ ID NO. 20.

[0400] In some preferred embodiments, the full-length sequence of human 2B4 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 21, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human 2B4 is shown in SEQ ID NO. 21.

[0401] In some preferred embodiments, the full-length sequence of human NTB-A comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 22, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NTB-A is as shown in SEQ ID NO. 22.

[0402] In some preferred embodiments, the full-length sequence of human CRACC comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 23, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human CRACC is shown in SEQ ID NO. 23.

[0403] In some preferred embodiments, the full-length sequence of human NKp80 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 24, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKp80 is as shown in SEQ ID NO.24.

[0404] CNK signal adapter components

[0405] In some embodiments, the CNK signal adapter component comprises at least (i) an NK cell signal converter (adaptor) (e.g., DAP10 or DAP12) or a functional variant thereof. In some embodiments, the NK cell signal converter comprises: (a) an NK cell signal converter extracellular domain (ED) or a functional variant thereof, (b) an NK cell signal converter transmembrane domain (TMD) or a functional variant thereof, and (c) an NK cell signal converter intracellular domain (ICD) or a functional variant thereof; optionally, a hinge or linker is included between the NK cell signal converter extracellular domain or a functional variant thereof, the NK cell signal converter transmembrane domain or a functional variant thereof, and / or the NK cell signal converter intracellular domain or a functional variant thereof.

[0406] In some preferred embodiments, the NK cell signal transducer is a NK cell signal transducer of mammalian origin; preferably, the mammal is selected from humans, primates, mice, horses, cows, sheep, goats, cats, pigs, dogs, llamas, alpacas, elephants, squirrels, and guinea pigs.

[0407] In some preferred embodiments, the NK cell signal transducer is a recombinant NK cell signal transducer comprising NK cell signal transducer domains from different sources.

[0408] In some preferred embodiments, the NK cell signal transducer is a human NK cell signal transducer; preferably, the NK cell signal transducer is a recombinant NK cell signal transducer comprising different human NK cell signal transducer domains.

[0409] In some preferred embodiments, the NK cell signal transducer is a murine NK cell signal transducer; preferably, the NK cell signal transducer is a recombinant NK cell signal transducer comprising different murine NK cell signal transducer domains.

[0410] In some preferred embodiments, the NK cell signal transducer is a recombinant NK cell signal transducer comprising human and murine NK cell signal transducer domains.

[0411] In some preferred embodiments, the extracellular domain of the NK cell signal transducer is the extracellular domain of a human or mouse NK cell signal transducer.

[0412] In some preferred embodiments, the transmembrane domain of the NK cell signal transducer is a transmembrane domain of a human or mouse NK cell signal transducer.

[0413] In some preferred embodiments, the intracellular domain of the NK cell signal transducer is the intracellular domain of a human or mouse NK cell signal transducer.

[0414] The NK cell signal converter in the CNK signal adapter component is DAP10 or DAP12.

[0415] In some preferred embodiments, the CNK cell signal transducer functional variant 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 mutant DAP10 or DAP12.

[0416] In some preferred embodiments, the CNK signal transduction component further comprises (ii) an immunoreceptor activation signaling domain (ITAM) and / or (iii) a T cell co-stimulatory signaling domain.

[0417] In some preferred embodiments, a hinge or linker is contained between the NK cell signal transducer or a functional variant thereof, the immunoreceptor activation signaling domain (ITAM) and / or the T cell co-stimulatory signaling domain; preferably, the NK cell signal transducer or a functional variant thereof is fused with the immunoreceptor activation signaling domain (ITAM) domain.

[0418] In some preferred embodiments, the immunoreceptor activation signaling domain (ITAM) is derived from the intracellular activation signaling domain of an immunoreceptor; preferably, the immunoreceptor 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 immunoreceptor activation signaling domain (ITAM) is fused to an NK cell signal transducer or a functional variant thereof; preferably, the immunoreceptor is CD3ζ;

[0419] In some preferred embodiments, the T cell costimulatory signaling domain is derived from the intracellular signaling domain of a costimulatory molecule.

[0420] In some preferred embodiments, the full-length sequence of human DAP10 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.25, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human DAP10 is as shown in SEQ ID NO.25.

[0421] In some preferred embodiments, the full-length sequence of human DAP10 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.26, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human DAP10 is as shown in SEQ ID NO.26.

[0422] In some preferred embodiments, the transmembrane domain of human DAP10 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 27, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the transmembrane domain of human DAP10 is as shown in SEQ ID NO. 27.

[0423] In some preferred embodiments, the full-length sequence of human DAP12 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.28, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human DAP12 is as shown in SEQ ID NO.28.

[0424] In some preferred embodiments, the transmembrane domain of human DAP12 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.29, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the transmembrane domain of human DAP12 is as shown in SEQ ID NO.29.

[0425] In some preferred embodiments, the transmembrane domain fusion protein of human DAP10 and human DAP12 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.30, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequences of the transmembrane domains of human DAP10 and human DAP12 are as shown in SEQ ID NO.30.

[0426] In some preferred embodiments, the human DAP10-DAP12 fusion protein sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.31, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP10-DAP12 fusion protein sequence is as shown in SEQ ID NO.31.

[0427] In some preferred embodiments, the human CD3zeta intracellular signaling domain sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.32, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human CD3zeta intracellular signaling domain sequence is as shown in SEQ ID NO.32.

[0428] In some preferred embodiments, the human DAP10-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.33, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP10-CD3zeta sequence is as shown in SEQ ID NO.33.

[0429] In some preferred embodiments, the human DAP12-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.34, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP12-CD3zeta sequence is as shown in SEQ ID NO.34.

[0430] In some preferred embodiments, the human DAP10-DAP12-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.35, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP10-DAP12-CD3zeta sequence is as shown in SEQ ID NO.35.

[0431] In some preferred embodiments, the human 41BB intracellular signaling domain sequence comprises an amino acid sequence having 80% or greater identity to the amino acid sequence shown in SEQ ID NO. 36, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity; the amino acid sequence of the human 41BB intracellular signaling domain sequence is shown in SEQ ID NO. 36.

[0432] In some preferred embodiments, the human DAP10-41BB sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown 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 99% or more identity; the amino acid sequence of the human DAP10-41BB sequence is as shown in SEQ ID NO.37.

[0433] In some preferred embodiments, the human DAP10-41BB-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.38, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP10-41BB-CD3zeta sequence is as shown in SEQ ID NO.38.

[0434] In some preferred embodiments, the human CD28 intracellular signaling domain sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 39, 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 99% or more identity; the amino acid sequence of the human CD28 intracellular signaling domain sequence is as shown in SEQ ID NO. 39.

[0435] In some preferred embodiments, the human DAP10-CD28 sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown 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 99% or more identity; the amino acid sequence of the human DAP10-CD28 sequence is as shown in SEQ ID NO.40.

[0436] In some preferred embodiments, the human DAP10-CD28-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.41, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP10-CD28-CD3zeta sequence is as shown in SEQ ID NO.41.

[0437] In some preferred embodiments, the human DAP12-41BB sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown 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 99% or more identity; the amino acid sequence of the human DAP12-41BB sequence is as shown in SEQ ID NO.42.

[0438] In some preferred embodiments, the human DAP12-41BB-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.43, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP12-41BB-CD3zeta sequence is as shown in SEQ ID NO.43.

[0439] In some preferred embodiments, the human DAP12-CD28 sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.44, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP12-CD28 sequence is as shown in SEQ ID NO.44.

[0440] In some preferred embodiments, the human DAP12-CD28-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.45, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP12-CD28-CD3zeta sequence is as shown in SEQ ID NO.45.

[0441] In some preferred embodiments, the CNK signal transduction component comprises an amino acid sequence selected from SEQ ID NOs. 25 to 45.

[0442] In some preferred embodiments, the chimeric protein construct forms a multifunctional complex with the NK cell activating receptor and the CNK signaling transduction component, 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 greater identity to the amino acid sequence set forth in SEQ ID NO. 117, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity; the amino acid sequence of the multifunctional complex comprises the amino acid sequence set forth in SEQ ID NO. 117.

[0443] 6. Nucleic acid molecules

[0444] In another aspect, the present disclosure also provides a chimeric nucleic acid construct encoding any chimeric protein construct described in the present application.

[0445] The nucleic acid molecule may include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with β-D-ribose configuration, α-LNA with α-L-ribose configuration (diastereomers of LNA), 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.

[0446] In some preferred embodiments, the nucleic acid molecule is mRNA. mRNA is any RNA encoding at least one protein, and the encoded protein can be produced in vitro, in vivo, in situ or in vitro. It will be understood by those skilled in the art that, unless otherwise stated, the nucleic acid sequence set forth in this application can be listed as "T" in a representative DNA sequence, but in the case where the sequence represents RNA (e.g., mRNA), "T" will be replaced with "U". Therefore, any DNA disclosed and identified herein by a specific sequence identification number also discloses a corresponding RNA (e.g., mRNA) sequence complementary to the DNA, wherein each "T" of the DNA sequence is replaced by a "U".

[0447] In some preferred embodiments, the nucleic acid molecule comprises a nucleotide sequence that is 80% or more identical to the nucleotide sequence shown in SEQ ID NO.118, preferably a nucleotide sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably a nucleotide sequence that is 98% or 99% or more identical.

[0448] In some preferred embodiments, the nucleic acid molecule comprises a nucleotide sequence that is 80% or more identical to the nucleotide sequence shown in SEQ ID NO. 160, preferably a nucleotide sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, more preferably a nucleotide sequence that is 98% or 99% or more identical. In some preferred embodiments, the nucleic acid molecule comprises a nucleotide sequence that is 80% or more identical to the nucleotide sequence shown in SEQ ID NO. 162, preferably a nucleotide sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, more preferably a nucleotide sequence that is 98% or 99% or more identical. In some preferred embodiments, the nucleic acid molecule comprises a nucleotide sequence that is 80% or more identical to the nucleotide sequence shown in SEQ ID NO. 165, preferably a nucleotide sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, more preferably a nucleotide sequence that is 98% or 99% or more identical.

[0449] 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, optionally including one or more modifications selected from the group consisting of: a 5'UTR, a 5'UTR, a poly A tail, a 5'-cap, and one or more modified nucleotides in the coding region.

[0450] i) Modification of nucleic acid molecules (e.g. mRNA)

[0451] Naturally occurring eukaryotic mRNA molecules may contain stabilizing elements, including but not limited to an untranslated region (UTR) at its 5'-end (5'UTR) and / or a UTR at its 3'-end (3'UTR), as well as other structural features, such as a 5'-cap structure, a 3'-poly A tail. Both the 5'UTR and the 3'UTR are typically transcribed from genomic DNA and are part of premature mRNA. The characteristic structures of mature mRNA (such as the 5'-cap structure and the 3'-poly A tail) are typically added to the transcribed (premature) mRNA during mRNA processing.

[0452] "5'UTR" refers to the region of an mRNA immediately upstream (i.e., 5') of the start codon (i.e., the first codon of the mRNA transcript translated by the ribosome) that does not encode a polypeptide. When an RNA transcript is generated, the 5'UTR may contain a promoter sequence. Such promoter sequences are known in the art. It is understood that such promoter sequences will not be present in the nucleic acid molecules of the present disclosure.

[0453] "3'UTR" refers to the region of an mRNA immediately downstream (ie, 3') of the stop codon (ie, the codon of an mRNA transcript that indicates the termination of translation) that does not encode a polypeptide.

[0454] A "poly A tail" is a downstream region of an mRNA, for example, immediately downstream (i.e., 3') of the 3' UTR, comprising a plurality of 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, the poly A tail contains 50 to 250 adenosine monophosphates. In a relevant biological context (e.g., in a cell or in vivo), the role of the poly A tail is to protect mRNA from enzymatic degradation (e.g., enzymatic degradation in the cytoplasm) and to facilitate transcription termination and / or export and translation of mRNA from the nucleus. The 3'-poly A tail is typically a section of adenine nucleotides added to the 3'-end of a transcribed mRNA. In some cases, it can contain up to about 400 adenine nucleotides. The length of the 3'-poly A tail can affect the stability of the mRNA molecule itself.

[0455] 5'-Capping of polynucleotides can be accomplished simultaneously during the in vitro transcription reaction using the following chemical RNA cap analogs 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 Biolabs, Ipswich, MA). 5'-Capping of modified RNA can be accomplished post-transcriptionally using vaccinia virus capping enzyme to generate a "Cap 0" structure: m7G(5')ppp(5')G (New England Biolabs, Ipswich, MA). Cap 1 structures can be generated using vaccinia virus capping enzyme and 2'-O methyltransferase: m7G(5')ppp(5')G-2'-O-methyl. The Cap 2 construct can be generated from the Cap 1 construct, followed by 2'-O-methylation of the 5'-antepenultimate nucleotide using a 2'-O methyltransferase. The Cap 3 construct can be generated from the Cap 2 construct, followed by 2'-O-methylation of the 5'-preantepenultimate nucleotide using a 2'-O methyltransferase. The enzyme can be derived from a recombinant source.

[0456] An mRNA molecule comprising at least one of the above-mentioned stabilizing elements can significantly increase the expression level of the protein it encodes, and when two or more of the above-mentioned stabilizing elements are used, it shows a synergistic effect in protein expression compared to mRNA using a single stabilizing element.

[0457] In some embodiments, the mRNAs described herein are depleted of one or more AU-rich sequences. AU-rich sequences, also known as AURES, are destabilizing sequences found in the 3' UTR.

[0458] In some embodiments, the mRNA described in the present disclosure further comprises an open reading frame (ORF) encoding a signal peptide. The signal peptide may comprise amino acids 15-60 at the N-terminus of the protein, which are typically required for transmembrane transport across the secretory pathway and, therefore, generally control the secretory pathway in which most proteins enter eukaryotes and prokaryotes. The length of 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.

[0459] An "open reading frame" is a continuous stretch of DNA that begins with a start codon (eg, methionine (ATG)) and ends with a stop codon (eg, TAA, TAG, or TGA) and encodes a polypeptide.

[0460] Signal peptides from heterologous genes are known in the art and can be tested for desired properties and then incorporated into the nucleic acids of the present disclosure. In some embodiments, the signal peptide can include one of the following sequences: 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).

[0461] The nucleic acid molecules provided herein can be synthesized by solid phase technology, liquid phase chemical synthesis, enzymatic ligation, and combinations thereof. The synthesized nucleic acid molecules can be purified and quantified. For details, see WO2021222304, the entire contents of which are incorporated herein by reference.

[0462] 7. Carrier

[0463] In another aspect, the present disclosure further provides a vector comprising a nucleic acid molecule as provided in the present disclosure, wherein the nucleic acid molecule is operably linked to at least one polynucleotide regulatory element (eg, a promoter) to express the chimeric protein construct encoded by the nucleic acid molecule.

[0464] 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.

[0465] In some embodiments, the viral vector is 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., malabsorption and sendai), 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 type 1 and 2, Epstein-Barr virus, cytomegalovirus) and poxvirus (e.g., vaccinia virus, fowlpox virus and canarypox virus), a Norwalk virus, a togavirus, a flavivirus, a reovirus, a papovavirus, a hepadnavirus, a baculovirus and a hepatitis virus, a virus-like particle (VLP).

[0466] In some embodiments, the viral vector is a retroviral vector.

[0467] In some embodiments, the retrovirus is selected from the group consisting of avian leukocytoblastoma, mammalian C-type, B-type, D-type viruses, HTLV-BLV collection, lentivirus, and foamy virus.

[0468] 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 can be inserted.

[0469] In some embodiments, the lentiviral vector is selected from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or ovine demyelinating leukoencephalitis lentivirus.

[0470] In some embodiments, the carrier is an expression vector based on a transposon. A transposon is a DNA sequence that can change its position within the genome. In a transposon system, the nucleic acid molecule flanking the chimeric protein construct provided herein is flanked by terminal repeats identifiable by a transposase that mediates transposon movement. The transposase can be co-delivered as a protein, encoded on the same carrier as the chimeric protein construct, or encoded on an independent carrier. Non-limiting examples of transposon systems include Sleeping Beauty, Piggyback, Frog Prince, and Prince Charming.

[0471] In some embodiments, the vectors provided by the present disclosure can be combined with other vectors comprising other nucleic acid molecules encoding at least one co-expression moiety.

[0472] In some preferred embodiments, the vector further comprises a promoter; preferably, the promoter is an EF1α promoter or a CMV promoter.

[0473] In some preferred embodiments, the chimeric protein construct provided by the present disclosure can be expressed in the same vector and the same promoter as the co-expression part, or under different promoters, or in multiple vectors.

[0474] 8. Cells

[0475] In another aspect, the present disclosure further provides an engineered cell, which expresses the chimeric protein construct provided by the present disclosure, or comprises the chimeric nucleic acid construct or vector provided by the present disclosure.

[0476] In some embodiments, the engineered cells simultaneously express the chimeric protein construct as provided in the present disclosure and the coexpression portion as described above. The coexpression portion and the targeting protein binding domain of the chimeric protein construct can be combined to achieve a specific purpose. For example, when the targeting protein binding domain specifically identifies the protein that can cause immune rejection expressed on immune cells, the coexpression portion can be a protein that further suppresses immune rejection (for example, viral ER resident glycoproteins, etc.), a protein that targets the tumor microenvironment (for example, chemokine receptors, etc.), a protein that stimulates immune activity (for example, cytokines, immune cell germline proteins CD7, CD5, etc.), and the resulting cells will have reduced immunogenicity (or immune rejection) and / or have enhanced immune activity when transplanted into the human body. This is very useful for cellular immunotherapy and other therapeutic cell transplants. Therefore, the cells can be immune cells (for example, T cells), stem cells, kidney cells, pancreatic islet cells, and cardiomyocytes, etc.

[0477] In some embodiments, the cell is an immune cell selected from the group consisting of: T cells, natural killer (NK) cells, B cells, macrophages, monocytes, dendritic cells, neutrophils or γδT cells. The T cells can 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, regulatory T cells, natural killer T cells, γ-δ T cells, cytokine-induced killer (CIK) T cells and tumor infiltrating lymphocytes.

[0478] In some embodiments, the engineered cells are T cells. In the case of expressing the chimeric protein constructs and co-expression moieties described above to obtain T cells with reduced immunogenicity and / or enhanced immune activity, the T cells may further express receptors that can specifically target tumor-associated markers, including but not limited to CAR, TCR, NK cell activating receptor components, etc.

[0479] In some embodiments, the engineered cells are T cells that simultaneously express a chimeric protein construct as provided herein and one or more selected from the group consisting of adenovirus E3-K19, CAR, NK cell activating receptor, and CNK signaling transduction component.

[0480] In some embodiments, the engineered cells are T cells that simultaneously express a chimeric protein construct as provided herein and a NK cell activating receptor and optionally a CNK signaling transduction component.

[0481] In some embodiments, the engineered cell is a T cell that simultaneously expresses a chimeric protein construct as provided in the present disclosure, adenovirus E3-K19, and CAR.

[0482] In another aspect, the present disclosure also provides a method for producing the engineered cells described herein (e.g., T cells), comprising introducing the vector described herein into a starting cell under conditions suitable for expressing the nucleic acid molecule described herein.

[0483] Many methods for producing CAR-T cells known in the art can also be applied to produce engineered cells as described herein. For example, Zhang et al., Engineering CAR-T cells, Biomarker Research (2017) 5: 22 describe methods for producing CAR-T cells. The methods provided herein may include one or more steps selected from the following: obtaining starting cells, culturing (including amplification, optionally including activation) starting cells, and genetically modifying 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 (HSC), CD34+ cells, embryonic cell line stem cells, mesenchymal stem cells, or iPSC cells. The starting cells can also be cells differentiated from stem cells, such as the immune cells described above.

[0484] The starting cells can be obtained from any source, for example, immune cells (e.g., T cells) that can be separated from a subject (e.g., a human subject). In some embodiments, immune cells are obtained from a subject of interest, such as a subject suspected of having a specific disease or condition, a subject suspected of having a susceptibility to a specific disease or condition, a subject who will experience, is experiencing, or has received treatment for a specific disease or condition. The subject can also be a healthy volunteer or a healthy donor; immune cells can also come from a blood bank. The immune cells can be autologous or allogeneic for the subject of interest. The immune cells can be collected from any location in the subject where they are present, including but not limited to blood, umbilical cord blood, spleen, thymus, lymph nodes, pleural effusion, splenic tissue, tumors, and bone marrow. The separated immune cells can be used directly or stored for a period of time, such as freezing.

[0485] The activation and / or expansion of immune cells is one of the main steps of immune cell function. In some embodiments, immune cells are activated and expanded while, before or / and after genetic modification. In some embodiments, immune cells are activated and / or expanded in vitro, in vitro or in vivo. The method of activating and expanding immune cells has been described in the art and can be used in the method described herein. For example, T cells can be activated and expanded by contacting with the surface of the ligand of the co-stimulatory molecule on the surface of the medicament and the stimulating CD3 / TCR complex related signal attached. In particular, T cell colonies can be stimulated, such as by contacting with anti-CD3 antibodies or their antigen-binding fragments or anti-CD2 antibodies fixed on the surface, or contacting with protein kinase C activators (such as bryostatin) and calcium ion carriers. In order to costimulate the auxiliary molecules on the surface of T cells, a ligand combined with the auxiliary molecule is used. For example, under conditions suitable for stimulating T cell proliferation, T cell colonies can be contacted with anti-CD3 antibodies and anti-CD28 antibodies. In order to stimulate the proliferation of CD4+T cells or CD8+T cells, anti-CD3 antibodies and anti-CD28 antibodies can be used. In certain embodiments, the primary stimulation signal and the co-stimulatory signal for T cells can be provided by different schemes.

[0486] Genetically modified cells can be achieved by transducing substantially homogeneous starting cells with nucleic acid molecules encoding chimeric protein constructs provided herein. In certain embodiments, nucleic acid molecules provided herein are introduced into starting cells using retroviral vectors (e.g., slow virus vectors). For example, nucleic acid molecules provided herein can be cloned into slow virus vectors, and expression can be driven from their endogenous promoters, slow virus long terminal repeats, or promoters specific to target cell types of interest. Common delivery methods for delivering viral vectors include, but are not limited to, electroporation, microinjection, gene guns, and magnetic transfection.

[0487] Genetically modified cells can also be achieved by delivering nucleic acid molecules with LNP, as specifically described in the section "6. Nucleic Acid Molecules" above. Other non-viral methods for nucleic acid delivery include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALE nucleases, CRISPR) can also be used to achieve genetic modification of the starting cells, thereby obtaining engineered cells as described in the present disclosure.

[0488] In certain embodiments, provided herein is an engineered cell by transfecting the nucleic acid molecules encoding the chimeric protein construct provided herein into starting cells before administration. In certain embodiments, provided herein is an engineered cell by being prepared by, for example, viral vector transfection immune cells with the nucleic acid molecules encoding the chimeric protein construct provided herein. Provided herein is an engineered cell showing reduced immunogenic molecules (for example, TCR, HLA, etc.) expression and / or immunosuppressive molecules (for example, PD-1, etc.) expression and / or enhanced tumor-targeted receptors (for example, CAR, engineered TCR, CNK receptors, etc.) expression on the cell surface.

[0489] On the other hand, the present disclosure also provides cell populations produced in vitro by the above methods. In certain embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the cell population express a detectable level of expression marker polypeptide provided herein (e.g., an EGFR truncate (EGFRt) that can be cleaved and connected to a chimeric protein construct as described herein). The expression level of the EGFR truncate can represent the expression level of the chimeric protein construct as described herein in the cell population.

[0490] 9. Pharmaceutical compositions and kits

[0491] In another aspect, the present disclosure further provides a pharmaceutical composition or a kit comprising (i) a chimeric protein construct, nucleic acid molecule, vector, or cell population as described herein, and (ii) a pharmaceutically acceptable vehicle. The term "pharmaceutically acceptable vehicle" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that can facilitate the storage and administration of the chimeric protein construct, nucleic acid molecule, expression vector, and / or cell population provided herein. Pharmaceutically acceptable vehicles can include any suitable component, such as, but not limited to, saline, liposomes, polymeric excipients, colloids, or carrier particles.

[0492] In certain embodiments, the pharmaceutically acceptable medium is a saline solution that can dissolve or disperse the chimeric protein constructs, nucleic acid molecules, expression vectors, and / or cell populations provided herein. Illustrative examples of saline solutions include, but are not limited to, buffered saline, sucrose solutions, physiological saline, acetate buffer, phosphate buffer, citrate buffer, bicarbonate buffer, saline solution, and polysorbate solution.

[0493] i) Nucleic acid pharmaceutical compositions

[0494] In certain embodiments, pharmaceutically acceptable medium is liposome.Liposome is unilamellar or multilamellar vesicle, has the film and internal aqueous part formed by lipophilic material.The nucleic acid molecule and / or carrier provided by the present disclosure can be encapsulated in the aqueous part of liposome.Exemplary liposome includes but is not limited to liposome based on 3 [N- (N ', N '-dimethylaminoethane) aminoformyl] cholesterol (DC-Chlo), based on N- (2,3- dioleoyloxy) propyl group-N, N, N- trimethylammonium chloride (DOTMA) liposome, and based on 1,2- dioleoyloxy -3- trimethylpropane (DOTAP) liposome.Prepare liposome and the method that nucleic acid molecule and / or carrier are encapsulated in liposome is well known in the art (referring to, for example, DDLasic et al, Liposomes in gene delivery, published by CRC Press, 1997).

[0495] In certain embodiments, the pharmaceutically acceptable medium is a polymeric excipient, including but not limited to microspheres, microcapsules, polymeric micelles and dendrimers. The nucleic acid molecules and / or vectors provided herein can be encapsulated, adhered or coated on a polymer-based component by methods known in the art (see, for example, W. Heiser, Nonviral gene transfer techniques, published by Humana Press, 2004; US patent 6025337; Advanced Drug Delivery Reviews, 57(15):2177-2202 (2005)).

[0496] In certain embodiments, the pharmaceutically acceptable medium is a colloid or carrier particle, such as gold colloid, gold nanoparticles, silica nanoparticles and multi-segment nanorods. The nucleic acid molecules and / or carriers provided herein can be coated, adhered to or combined with a carrier in any suitable manner known in the art (see, for example, 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)).

[0497] In certain embodiments, the pharmaceutical composition may further comprise additives, including but not limited to stabilizers, preservatives, and transfection facilitators that facilitate cellular uptake of the drug. Suitable stabilizers may include but are not limited to sodium glutamate, glycine, EDTA, and albumin. Suitable preservatives may include but are not limited to 2-phenoxyethanol, sodium benzoate, potassium sorbate, methyl hydroxybenzoate, phenols, thimerosal, and antibiotics. Suitable transfection facilitators may include but are not limited to calcium ions.

[0498] 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 route of administration. Preparations suitable for pharmaceutical composition administration can include but not limited to solutions, dispersions, emulsions, powders, suspensions, aerosols, sprays, nasal drops, liposome-based formulations, patches, implants and suppositories.

[0499] The formulations can be conveniently presented in unit dosage form and can be prepared by any method well known in the pharmaceutical art. The method of preparing these formulations or pharmaceutical compositions comprises the step of providing the nucleic acid molecules described herein to one or more pharmaceutically acceptable vehicles and optionally one or more additives. Methods for preparing such formulations can be found in, for example, Remington's Pharmaceutical Sciences (Remington: The Science and Practice of Pharmacy, 19th ed., A. R. Gennaro (ed), Mack Publishing Co., NJ, 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);, which reference is incorporated herein by reference in its entirety).

[0500] In some embodiments, the nucleic acid molecule (eg, mRNA) can be delivered physically, biologically, or chemically (see, e.g., S. Guan, J. Rosenecker, Gene Ther. 2017, 24, 133.).

[0501] Physical methods include, but are not limited to, delivery by gene gun (e.g., a gene gun using Au-particles), electroporation, sonoporation, and the like (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.).

[0502] Biological methods include, but are not limited to, delivery via viral vectors (eg, retroviral vectors, adenoviral vectors, adeno-associated viral vectors).

[0503] Chemical methods include, but are not limited to, delivery via natural proteins / polysaccharides, polymers, and lipids. Exemplary natural proteins / polysaccharides include protamine and chitosan (see, e.g., AE 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).

[0504] Exemplary lipids include cationic lipids such as 1,2-diocta-decenyl-3-trimethy lammonium-propane (DOTMA) and 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) (see, e.g., X. Hou et al., Nat. Rev. Mater. 2021, 10, 1078.), liposomes formed by DOTMA, DOTAP and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, helper lipid), which can form colloid-stabilized nanoparticles after self-assembly with mRNA (see, e.g., L. M. Kranz et al., Nature 2016, 534, 396.).

[0505] Exemplary lipids can also be ionizable lipids. Ionizable lipids (pKa 6.5-6.9) are alternative lipid materials, neutral at physiological pH, but positively charged by the protonation of free amines in an acidic environment (see, for example, SC Semple et al., Nat. Biotechnol. 2010, 28, 172). After cell internalization, the nanoparticles formed by ionizable lipids are encapsulated in endosomes (endosomes). Subsequently, due to the continuous decline of pH value in endosomes and lysosomes, ionizable lipids obtain protons for ionization, thereby promoting the fusion of lipid nanoparticles (lipid nanoparticle, LNP) with endosomal membranes, ultimately causing the mRNA loaded on lipid nanoparticles to be released into cytoplasm (see, for example, L. Miao et al., Mol. Cancer 2021, 20, 41.).

[0506] Ionizable lipids can be combined with cholesterol, helper lipids, and PEGylated lipids (i.e., PEGylated lipids) to form lipid nanoparticle formulations. Cholesterol is a naturally rigid and hydrophobic lipid that maintains the structure and stability of lipid nanoparticles. It can also promote the fusion of mRNA-loaded lipid nanoparticles (i.e., mRNA nanoparticles) with endosomal membranes. Helper lipids (such as zwitterionic lipids DOPE, 1,2-distearoyl-snglycero-3-phosphocholine (DSPC) and 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)) are widely used to promote cell membrane penetration and endosomal membrane escape (see, for example, N. Chaudhary et al., Nat. Rev. Drug Discovery 2021, 20, 817.). PEGylated lipids are composed of PEG and anchor lipids. The hydrophilic PEG is mainly distributed on the surface of the mRNA complex, while the hydrophobic region is embedded in the interior of the lipid bilayer. The introduction of PEGylated lipids not only increases the half-life of lipid nanoparticles but also can regulate the particle size by changing the molecular weight of the PEG chain. Typically, the molecular weight and lipid tail length can range 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.).

[0507] Over the past few decades, researchers have developed a large library of ionizable lipids for mRNA delivery, including DLin-MC3-DMA, SM-102, TT3, C12-200, 306O i10and ALC-0315 (see, for example, 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 them have achieved remarkable results in clinical applications. A typical example is DLin-MC3-DMA, which is a key component of Onpattro approved by the U.S. Food and Drug Administration (FDA) for siRNA delivery (see, for example, A.Akinc et al., Nat.Nanotechnol.2019,14,1084). DLin-MC3-DMA is also widely used in mRNA delivery, including protein and peptide replacement, gene editing and antiviral infection (see, for example, RSRiley et al., Sci.Adv.2021,7,eaba1028.). SM-102 and ALC-0315, two "star molecules", have been approved by the FDA as key components in BNT162b and mRNA-1273 vaccines for preventing COVID-19, respectively (see, for example, X.Hou et al., Nat.Rev.Mater.2021,10,1078.).

[0508] An ideal lipid-based mRNA carrier must meet the following conditions: 1) naked mRNA can form a stable complex to protect it from degradation; 2) four key components (ionizable lipids, cholesterol, helper lipids, and PEGylated lipids) should be added to stabilize the mRNA complex; 3) the components of the lipid nanoparticles should be protonated to induce membrane destabilization and promote endosomal escape of the mRNA complex; and 4) all lipid materials are biodegradable and do not cause any harm to patients.

[0509] The following points should be considered when optimizing lipid-based delivery platforms: 1) Degradability of ionizable lipids: The backbone structure of lipids promotes lipid clearance and reduces toxicity by introducing alkyne and ester groups into the lipid tail; 2) Immunogenicity of lipid nanoparticles: Heterocyclic lipids within lipid nanoparticles can improve the efficiency of mRNA vaccines by activating the interferon gene stimulator (STING) pathway of dendritic cells (DCs) (see, for example, L. Miao et al., Nat. Biotechnol. 2019, 37, 1174.); 3) Stability of lipid nanoparticles: Some promising strategies are expected to improve the stability of mRNA vaccines, including pK a optimization, excipient introduction and mRNA modification, etc.

[0510] Lipid nanoparticles can be produced using components, compositions, and methods as are known in the art, see, for example, PCT / US2016 / 052352, PCT / US2016 / 068300, PCT / US2017 / 037551, PCT / US2015 / 027400, PCT / US2016 / 047406, PCT / US2016 / 000129, PCT / US2016 / 01428 ...7 / 0 6 / 014280, PCT / US2017 / 038426, PCT / US2014 / 027077, PCT / US2014 / 055394, PCT / US2016 / 052117, PCT / US2012 / 069610, PCT / US2017 / 027492, PCT / US2016 / 059575, and PCT / US2016 / 069491, the entire contents of which are incorporated herein by reference.

[0511] ii) Oncolytic viruses and pharmaceutical compositions thereof

[0512] In some embodiments, the present application provides an oncolytic virus capable of expressing the chimeric protein construct described herein. In this application, "oncolytic virus" refers to any virus that can infect tumor cells, replicate in tumor cells, and dissolve tumor cells. In certain embodiments, the oncolytic virus is further capable of spreading to other tumor cells in a continuous replication cycle.

[0513] Oncolytic viruses can be derived from a variety of viruses, non-limiting examples of which include vaccinia 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) and Newcastle disease virus (NDV), as well as variants of these viruses.

[0514] In some embodiments, the present application provides an oncolytic virus comprising a nucleic acid molecule as provided herein, which is operably linked to at least one polynucleotide regulatory element (e.g., a promoter) to express a chimeric protein construct encoded by the nucleic acid molecule.

[0515] In some embodiments, the oncolytic viruses described herein can infect substantially 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.

[0516] It is understood that oncolytic viruses can be made replication selective if viral replication is under the control of gene expression regulators, such as the enhancer / promoter region derived from the 5' side of the albumin gene (see, for example, Miyatake et al., 1997, J. Virol. 71: 5124-5132).

[0517] In some embodiments, the oncolytic virus may have a tumor cell-specific promoter or a tumor cell-specific transcriptional regulatory sequence in its genome. "Tumor cell specificity" for a promoter or transcriptional regulatory sequence means that it is typically present in target tumor cells at a higher level than normal cells. In this way, an enhanced level of tumor cell specificity can be conferred oncolytic viruses.

[0518] For example, by operably linking the HSV gene to the TGF-β promoter, the main transcription unit of HSV can be placed under the transcriptional control of the tumor growth factor-β (TGF-β) promoter. It is known that certain tumor cells overexpress TGF-β relative to non-tumor cells of the same type. Therefore, an oncolytic virus in which replication is subject to the transcriptional control of the TGF-β promoter is replication selective because it is more capable of replicating in certain tumor cells than in non-tumor cells of the same type. Similar replication selective oncolytic viruses can be prepared using any gene expression regulator known to selectively cause overexpression in affected cells. Replication selective oncolytic viruses can be, for example, HSV-1 mutants in which the gene encoding ICP34.5 is mutated or deleted. Oncolytic viruses can also further include other modifications in their genome. For example, an oncolytic virus can include additional DNA inserted into the UL44 gene. This insertion can produce functional inactivation of the UL44 gene and the resulting lysis phenotype, or it can be inserted into an already inactivated gene, or replace a deleted gene.

[0519] In some embodiments, the oncolytic virus is engineered to place the nucleic acid sequence encoding the chimeric protein construct described herein under the control of a tumor cell-specific promoter.

[0520] In some embodiments, the oncolytic virus is engineered to place a gene encoding at least one protein required for viral replication under the control of a tumor cell-specific promoter.

[0521] In some embodiments, the oncolytic virus vector can express the chimeric protein construct described herein, and the targeting domain of the chimeric protein construct can recognize and bind to tumor proteins. Tumor proteins can be proteins highly expressed in tumor cells (e.g., c-Myc, Bcl-2, Bcl-xL, Bcl-w, KRAS), proteins expressed in tumor cells that promote tumor cell growth (e.g., estrogen receptor, androgen receptor, Her2, VEGF, VEGFR, PDGFRβ, EGFR, EGFR mutants), or proteins that promote tumor cells to escape normal immune responses (e.g., PD-L1, TGF-β1).

[0522] In some embodiments, the targeting domain of the chimeric protein construct can bind to Bcl-2. Any targeting domain capable of targeting Bcl-2 can be used, for example, but not limited to, the development of nanoantibody sequences targeting the highly conserved and homologous BH1, BH2 motifs of Bcl-2 anti-apoptotic family members can be used as targeting domains. After the ER-TPD element targeting Bcl-2 family proteins is constructed and cloned and loaded onto a strong oncolytic virus promoter, after specific infection of tumor cells, the oncolytic virus is amplified in large quantities and expresses the ER-TPD element of the Bcl-2 family protein, thereby achieving degradation of the intracellular Bcl-2 anti-apoptotic member proteins and directly leading to tumor cell apoptosis.

[0523] In some embodiments, the targeting domain of the chimeric protein construct can bind to VEGFR. Any targeting domain capable of targeting VEGFR can be used, for example, but not limited to, VEGFR antibodies (anti-VEGF-A antibody Bevacizumab and anti-VEGFR2 antibody Ramucizumab,) heavy chain and light chain recombinant scFv series ER-TPD element, constructing a targeting VEGFR ER-TPD element, and cloning and loading it on a strong oncolytic virus promoter. After specific infection of tumor cells, the oncolytic virus is amplified in large quantities and expresses the VEGFR ER-TPD element, directly inhibiting the expression of VEGFR in tumor cells, resulting in growth inhibition and apoptosis of tumor cells due to lack of sufficient nutrients.

[0524] In some embodiments, the targeting domain of the chimeric protein construct can bind to TGF-β1 and PD-L1. Any targeting domain that can target and bind to bcl-2 can be used, for example, but not limited to, using the heavy chain and light chain recombinant scFv of TGF-β1 and PD-L1 antibodies in series with ER-TPD elements to construct TGF-β1 and PD-L1ER-TPD elements, and cloning and loading them on the strong promoter of oncolytic virus. After specific infection of tumor cells, the oncolytic virus is amplified in large quantities and expresses TGF-β1 and PD-L1ER-TPD elements, directly inhibiting and degrading the expression of TGF-β1 and PD-L1 in tumor cells, breaking the tumor immunosuppressive microenvironment, and thus achieving the effect of treating tumors.

[0525] Without being limited by theory, it is believed that the chimeric protein construct of the present application has unique advantages when expressed in tumor cells via an oncolytic viral vector. On the one hand, the chimeric protein construct of the present application can target and degrade proteins in tumor cells that help tumor cells grow or escape immune system or resist apoptosis, thereby promoting apoptosis or death of tumor cells. On the other hand, the oncolytic viral vector can specifically replicate in tumor cells, promoting tumor cell lysis through viral replication and lysis. By integrating two different tumor-killing methods into the oncolytic viral vector, a synergistic killing effect can be achieved in the same drug form.

[0526] In another aspect, the present disclosure further provides a pharmaceutical composition comprising an oncolytic virus provided herein and a pharmaceutically acceptable medium. In certain embodiments, the pharmaceutically acceptable medium comprises any and all transport vehicles, solvents, diluents, excipients, adjuvants, dispersion media, coatings, antibacterial and antifungal agents, absorbents, and the like that are compatible with administration in mammals, and particularly human subjects.

[0527] In certain embodiments, the oncolytic virus or its pharmaceutical composition described herein is formulated for intravenous or intratumoral administration. In certain embodiments, the oncolytic virus or its pharmaceutical composition may be placed in a solvent or diluent suitable for human or animal use. The solvent or diluent is preferably isotonic, hypotonic or weakly hypotonic and has a relatively low ionic strength. Typical examples include sterile water, normal saline (such as sodium chloride), Ringer's solution, glucose, trehalose or sucrose solution, Hank's solution and other aqueous physiologically balanced salt solutions.

[0528] In one embodiment, the oncolytic virus or its pharmaceutical composition described herein may have 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 or slightly alkaline pH (e.g., from about pH 7 to about pH 9).

[0529] In one embodiment, the oncolytic virus composition of the present invention may be prepared to improve its stability, especially under production conditions and for long-term (ie, at least 6 months, preferably at least 2 years) frozen (eg, -70 ° C, -20 ° C) storage, refrigerated storage (eg, 4 ° C), and room temperature storage. A variety of existing viral preparations in the art are in either frozen liquid form or in freeze-dried form (eg, WO98 / 02522 and WO2008 / 114021, etc.). A solid (eg, dry powder or freeze-dried) composition can be obtained by steps involving vacuum drying and freeze-drying. For illustrative purposes, a buffered formulation with the addition of NaCl and sugar is particularly suitable for preserving viruses.

[0530] iii) Cellular pharmaceutical compositions

[0531] In another aspect, the present disclosure also provides a cell pharmaceutical composition comprising the engineered cells or cell populations provided by the present disclosure and a pharmaceutically acceptable medium. Exemplary media include buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides 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 cell pharmaceutical composition of the present invention is formulated for intravenous administration.

[0532] III. Application of TPD

[0533] In one aspect, the present disclosure further provides a use of the chimeric protein construct, nucleic acid molecule, or vector described herein in preparing a cell for treatment. In one aspect, the present disclosure further provides a use of the chimeric protein construct, nucleic acid molecule, or vector described herein in treating a disease. In one aspect, the present disclosure further provides a use of a cell expressing the chimeric protein construct, nucleic acid molecule, or vector described herein in treating a disease.

[0534] 1. Cell Transplantation

[0535] In one aspect, the present disclosure also provides a use of the chimeric protein construct, nucleic acid molecule or vector described herein in preparing cells to be transplanted (eg, allogeneic cell transplantation).

[0536] In certain embodiments, the cells to be transplanted are immune cells (e.g., T cells), stem cells (and cells differentiated therefrom), kidney cells, pancreatic islet cells, cardiomyocytes, etc. In certain embodiments, the cells to be transplanted can be autologous cells or allogeneic cells.

[0537] On the one hand, the application also provides a cell to be transplanted that expresses the chimeric protein construct, nucleic acid molecule or vector described above, wherein the chimeric protein construct described in the application is expressed, and the chimeric protein construct comprises an ERAD mechanism protein binding domain and a targeting domain, and the targeting domain comprises a domain that specifically targets or binds to a target protein, and the target protein is a transplant rejection-related protein. In certain embodiments, the chimeric protein construct further comprises a protein degradation pathway member (e.g., E3 ubiquitin ligase, proteasome, lysosome) binding domain as described in the application. Any protein degradation pathway member binding domain described in the application can be used.

[0538] In certain embodiments, the transplant rejection reaction-related proteins include antigen presenting molecules (such as MHC class I molecules, MHC class II molecules, MICA / B molecules, etc.), antigen recognition molecules (such as TCR, CD123, NKG2D, etc.), immune checkpoint molecules (such as PD-1, PD-L1, CTLA4, TIM3, TIGIT, LAG3, A2AR, BTLA, IDO1, IDO2, TDO, KIR, NOX2, VISTA, SIGLEC7, PVR, etc.), etc.

[0539] In certain embodiments, the transplant rejection-related protein comprises an immunogenic protein (eg, HLA (HLAα / β), TCR (αβTCR), NKG2D (natural killer cell group 2 member D) ligand, etc.).

[0540] The present disclosure also provides the above-mentioned cells to be transplanted and treatment methods thereof, which can overcome the problems faced in existing cell transplantation and cell therapy, such as the source of personalized transplanted cells and therapeutic cells, the inability to standardize and scale production, and low treatment efficiency.

[0541] The immune system has powerful plasticity and a nearly unlimited ability to detect invading viruses, bacteria, foreign cells, and diseased cells. This extraordinary immune surveillance capability is primarily achieved through humoral and cellular immunity, which include two important molecular structures: immunoglobulins and T cell receptors (TCRs). The TCR is the defining structure of T cells and is a transmembrane heterodimer composed of α and β chains or δ and γ chains connected by disulfide bonds. Within these chains, the complementarity determining regions (CDRs) determine the antigens that the TCR will bind to. Within 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.

[0542] The human MHC class I gene region includes alleles at the HLA-A, B, and C loci, encoding classic class I antigens (molecules), such as HLA-A, B, and C antigens, known as HLA-A, HLA-B, and HLA-C. These antigen molecules are present on the surface of all somatic cells and bind to intracellular protein epitope peptides for recognition by the immune system. If a cell produces a mutant protein or is invaded by foreign bacteria or viruses, the cell presents these mutant proteins or heterologous protein epitopes. Immune cells recognize these mutant proteins or heterologous protein epitopes and initiate an immune attack and killing, thereby eliminating the diseased cells, pathogens, and viruses that invade the cells.

[0543] 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 αβ TCR dimer that binds antigen and three invariant dimers (CD3γε, δε, and ζζ) that participate in TCR.CD3 surface trafficking, stabilization, and signal transduction. The αβ T cell receptor (αβ TCR) is expressed on the majority (approximately 95%) of 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 pathogenesis of graft-versus-host disease (GVHD) during allogeneic hematopoietic cell transplantation (allo-HCT) and allogeneic CAR-T cell therapy.

[0544] 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 responsible for regulating the immune system. In the therapeutic transplantation setting, "HLA mismatch" occurs when the donor HLA on the allograft differs from that of the recipient. HLA mismatch leads to the activation of alloreactive T cells, which can cause acute cellular rejection (ACR) within six months of transplantation. Mismatched donor HLA antigens are also targets for the development of de novo donor-specific HLA antibodies (dnDSA), which play a reinforcing role in acute and chronic rejection of transplanted cells (such as T cells). Therefore, to generate universal transplantable cells (such as T cells) for safe allogeneic infusion and therapeutic purposes, effective blockade of graft-versus-host disease (GVHD) by genetically disrupting the TCR is recommended. In addition, it is necessary to suppress HLA expression on allogeneic cells (such as T cells) to reduce the recipient's immune system's rejection of the allogeneic T cell TCRαβ and / or HLA class I.

[0545] In certain embodiments, the cells to be transplanted that express the chimeric protein constructs, nucleic acid molecules, or vectors described above have reduced immunogenicity.

[0546] In some embodiments, the cells to be transplanted with reduced immunogenicity (or with high compatibility) described in the present application are T cells, which express a TPD chimeric protein construct comprising an scFv that specifically recognizes TCR and / or HLA and a transmembrane domain or a functional variant thereof and an endoplasmic reticulum retention domain or a functional variant thereof of the HCMV glycoprotein US2 and / or US11.

[0547] The ERAD mechanism protein binding domain comprises an amino acid sequence as shown 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.

[0548] In some embodiments, the cells to be transplanted with reduced immunogenicity are T cells, which express an ER-TPD chimeric protein construct comprising a scFv that specifically recognizes TCR and a transmembrane domain of adenovirus E3-K19 or a functional variant thereof and an endoplasmic reticulum retention domain or a functional variant thereof. In some embodiments, the cells to be transplanted with reduced immunogenicity are T cells, which express an ER-TPD chimeric protein construct comprising a targeting protein binding domain and an ERAD mechanism protein binding domain, wherein the targeting protein binding domain comprises 6 CDRs comprising an amino acid sequence as shown 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 mechanism protein binding domain comprises an amino acid sequence as shown 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.

[0549] In some embodiments, the above-mentioned T cell-expressed ER-TPD chimeric protein construct with reduced immunogenicity further comprises a protein degradation pathway member (e.g., E3 ubiquitin ligase, proteasome) binding domain, optionally, the protein degradation pathway member binding domain is connected to the ERAD mechanism protein binding domain. In some embodiments, the above-mentioned T cell-expressed ER-TPD chimeric protein construct with reduced immunogenicity further comprises an E3 ubiquitin ligase binding domain having an amino acid sequence as shown in SEQ ID NO.147.

[0550] In some embodiments, the cells to be transplanted (such as T cells) with reduced immunogenicity further express the co-expression moiety as described above.

[0551] For example, in order to reduce the immunogenicity or antigen presentation of cells, proteins that can degrade or reduce MHC class I or class II molecules can be expressed as co-expressed parts in the above-mentioned cells to be transplanted (such as T cells) with reduced immunogenicity (for example, complete viral ER-resident glycoproteins, including but not limited to HCMV US2, US3, US11, US10, adenovirus E3-K19, HCMV US6 and HSV ICP47). Effective downregulation of TCR will greatly inhibit TCR-mediated immune attack and reduce GVHD during allogeneic T cell infusion. Glycoproteins including natural viral ER-resident can further inhibit MHC molecules, thereby preventing peptides from being presented to recipient CD8+ T cells and inhibiting the immune recognition of allogeneic T cells. Therefore, the highly compatible T cells described above are also called "autologous universal T cells (UT)" or "universal T cells (UT)", which can achieve the purpose of allogeneic transfusion therapy and can improve the compatibility of allogeneic T cells and the long-term persistence after infusion.

[0552] On the other hand, the present disclosure provides a method for reducing the subject's immune response to cell transplantation, comprising administering to the subject a therapeutically effective amount of the cells to be transplanted as described above with reduced immunogenicity, thereby reducing the subject's immune response to the transplant or to the modified cells. In certain embodiments, the cells to be transplanted express a TPD chimeric protein construct as described herein in the cell, the TPD chimeric protein construct comprising a domain targeting immunogenic protein (e.g., HLA (HLA α / β), TCR (αβTCR), NKG2D (natural killer cell 2 family member D) ligand, etc.) and the ERAD mechanism protein binding domain described above, and optionally, a protein degradation pathway member binding domain.

[0553] 2. Cell Therapy

[0554] Cell therapy involves genetically engineering cells (such as immune cells) to present tumor-associated antigens or express receptors that specifically recognize diseased cells. These cells are then exponentially expanded in vitro and then infused back into the patient's body, activating the immune system to attack tumor cells or directly and specifically recognize and kill them. In 2012, CD19-targeted CAR-T cells achieved the first-ever targeted elimination of tumor cells in a B-cell leukemia patient, becoming the next truly curative leukemia treatment after bone marrow stem cell transplantation and ushering in a new era of precision medicine cell therapy. This technology has the potential to be applied to the treatment of various hematologic malignancies and solid tumors. However, the current clinical efficacy of conventional CAR-T in treating solid tumors is poor. The reasons are: (1) The killing function of CAR-T is highly dependent on the recognition of tumor-associated antigens (TAA) by the CAR structure, but due to the heterogeneity of solid tumors, there are great differences in the expression of target proteins on the surface of tumor cells; currently, single-target CAR-T cannot completely recognize and kill all tumor cells, which leads to tumor immune escape and recurrence and metastasis; (2) The direct immunosuppressive effect of solid tumors, expressing PD-L1, B3H7, etc., directly inhibits the activation of T cells; (3) The immunosuppressive microenvironment of solid tumors contains a large number of immunosuppressive cells, such as TAM, Treg, and MDSC. It is difficult for conventional CAR-T to break through the limitations of immunosuppressive cells inside the tumor; (4) Conventional CAR-T is a personalized cell therapy. The T cells come from the patient himself. If the patient has received a lot of radiotherapy and chemotherapy, and the immune system function is damaged, it is difficult to isolate a sufficient number of T cells from the patient's peripheral blood. Even if they are isolated and transformed, the proliferation and killing function of T cells are still very weak, so it is difficult to achieve a therapeutic effect.

[0555] The cells to be transplanted (eg, T cells) provided in this application can overcome the problems faced in existing cell therapy, such as differences in tumor targets, tumor immunosuppressive environment, and sources of personalized therapeutic cells.

[0556] For the purpose of cell therapy, the cells to be transplanted (e.g., T cells, further, universal T cells with defects in TCRαβ and MHC molecule surface expression) with reduced immunogenicity can be further genetically engineered, for example, to express the co-expression portion as described above, to achieve therapeutic purposes.

[0557] For example, in order to enable the cells to be transplanted (such as T cells) to specifically act on disease-related target proteins or cells expressing disease-related target proteins, binding domains or molecules that recognize or bind to disease-related target proteins can be expressed in the above-mentioned cells to be transplanted (such as T cells) with reduced immunogenicity, such as chimeric antigen receptors CAR, engineered TCR, or CNK receptors provided in this application, or antiviral protein binding domains, etc.

[0558] In some embodiments, molecules (such as chemokine receptors or cytokine receptors) that can mediate the migration of the transplanted disease to the disease microenvironment can also be expressed in the above-mentioned cells to be transplanted (such as T cells) with reduced immunogenicity.

[0559] For another example, in order to increase the immunostimulatory activity of cells to be transplanted (such as T cells), immunostimulatory molecules can be expressed in the above-mentioned cells to be transplanted (such as T cells) with reduced immunogenicity.

[0560] On the other hand, the present application also provides a method for introducing NK elements, especially optimized recombinant NK elements, into the cells to be transplanted (such as UT cells) provided in the present application, so that T cells can recognize all virus-infected cells and tumor cells as efficiently and broadly as NK cells. Optionally, by further optimizing the transduction elements, CNK-T cells can be efficiently activated and kill tumor cells. Because NK targets include family member proteins such as MICA, MICB and ULBP1-6, which can be widely expressed in various types of tumor cells and cover different stages of tumor progression, CNK technology can effectively solve the off-target effects of single CAR-T and eliminate the chance of tumor immune escape. At the same time, the composite adapter (Chimeric Adaptor) introduced by CNK in the design can effectively transduce and amplify NK signals, transcend the limitations of immune checkpoints such as PD1 signals, efficiently activate T cells, and achieve the killing of tumor cells. The Chimeric Adapter amplifies CNK-T cell signals, counteracting immunosuppression in the tumor environment and activating T cells to kill tumor cells. Furthermore, CNK-T cells, through NK recognition of targets, can eliminate immunosuppressive cells such as MDSCs. After viral infection, cells express specific functional proteins that stimulate the assembly or transport of MHCI or directly promote the targeted degradation of MHCI molecules, thereby inhibiting the presentation of viral antigen epitopes and promoting immune escape.

[0561] On the one hand, the present disclosure provides a UT cell that also expresses a CNK receptor and, optionally, a CAR. Such UT cells may also be referred to as "CNKT-UT." In this application, it is verified that CNK-UT has a broad spectrum of tumor recognition and killing capabilities. This application further designs a composite specific target CAR / CNK-UT product, demonstrating that the CAR / CNK-UT product has a more powerful killing and activation function on tumor cells than conventional CAR-T; in animal experiments, the CAR / CNK-UT product also has a more efficient tumor clearance ability. UT technology realizes the transformation of allogeneic universality, and T cells are derived from healthy donors, thereby realizing the standardization and large-scale production of CNK-UT products, which can be prepared in advance and ensure the function of T cells to kill and activate tumor cells.

[0562] In some preferred embodiments, the CNKT-UT comprises a nucleic acid molecule encoding an amino acid sequence having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO. 117, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity. 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 also provides the use of CNKT-UT in the preparation of a medicament for treating a disease.

[0563] On the one hand, the present disclosure further provides a kind of comprising or expressing the chimeric protein construct described in the present application, encoding its nucleic acid molecule or the immune cell of carrier, wherein the chimeric protein construct includes ERAD mechanism protein binding domain and targeting domain, and the targeting domain includes specific targeting or binding domain of target protein, and the target protein is expressed on the immune cell and on the target cell. In certain embodiments, the immune cell further includes or expresses the binding domain or molecule (such as CAR) targeting the target protein, encoding its nucleic acid molecule or carrier. In certain embodiments, examples of such target proteins include CD123, CD5, CD7, CD38, or CD4.

[0564] Without being limited by theory, some target proteins are expressed both on target cells (such as cancer cells or virus-infected cells) and on immune cells for treatment. Such targets are usually difficult to treat by cell therapy methods, so when CAR targeting such targets is expressed on immune cells for treatment, CAR will also lead to the same target expressed by the immune cells themselves, causing autophagy in the immune cells, and then unable to amplify normally, or having limited effect on target cell killing. By the chimeric protein construct provided by the application, the target protein expressed by the immune cells themselves can be degraded, thereby avoiding autophagy, while retaining the specific killing effect on the target cells. Another advantage of using the chimeric protein construct of the application is that the chimeric protein construct targeting the target and the binding domain targeting the target (such as CAR, TCR, etc.) can be expressed simultaneously by co-expression, which is simpler and more effective than methods such as traditional gene knockout.

[0565] In certain embodiments, the chimeric protein construct further comprises a protein degradation pathway member (e.g., E3 ubiquitin ligase, proteasome, lysosome) binding domain as described herein. Any protein degradation pathway member binding domain described herein can be used.

[0566] Methods of administering cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, methods for adoptive T cell therapy are described in, for example, 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.

[0567] In certain embodiments, cells or individual populations or subtypes of cells are administered to a subject in the 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 foregoing 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 foregoing values), or about 100 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 foregoing values). In some embodiments, the number of cells present in the cell culture medium is about 10 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, about 65 billion 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 foregoing 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.

[0568] In some embodiments, the dose of total cells and / or the dose of individual subpopulations of cells is at or about 10 4 cells / kg body weight to at or around 10 9 cells / kg body weight, for example, in the range of 10 5 and 10 6 cells / kg body weight, for example, at least or at least about or at or about 1×10 5 cells / kg, 1.5×10 5 cells / kg, 2×105 cells / kg or 1×10 6 cells / kg body weight. For example, in some embodiments, the cells are expressed at or about 10 4 and at or about 10 9 T cells / kg body weight or within a certain error range thereof, for example, between 10 5 and 10 6 T cells / kg body weight, such as at least or at least about or at or about 1×10 5 T cells / kg, 1.5×10 5 T cells / kg, 2×10 5 T cells / kg, or 1×10 6 T cells / kg body weight.

[0569] Cell can be used by any suitable mode, for example, by bolus, by injection, for example intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, through septal injection, subscleral injection, intrachoroidal injection, intracameral injection, injection under perineum, subconjunctival injection, under Tenon's fascia capsule (sub-Tenon) injection, retrobulbar injection, peribulbar injection, or posterior sclera (posterior juxtascleral) delivery. In some embodiments, they are used by parenteral, intrapulmonary and intranasal administration, and if local treatment is desired, then by intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In some embodiments, given dose is used by single bolus administration cell to use. In some embodiments, it is used by repeatedly bolus administration cell to use, for example, during being no more than 3 days, or by continuous infusion administration cell to use.

[0570] In some embodiments, a repeated dose method is provided, in which a first dose of cells is administered, followed by one or more second continuous doses. When administered to a subject using an adoptive therapy approach, the timing and size of multiple doses of cells are typically designed to increase the efficacy and / or activity and / or function of T cells expressing antigens (e.g., T cells expressing CAR). In some embodiments, repeated administration reduces the downregulation or inhibitory activity that may occur when inhibitory immune molecules such as PD-1 and / or PD-L1 are upregulated on T cells expressing antigens, such as CARs. The method includes administering a first dose, typically followed by one or more continuous doses, and having a specific time frame between different doses.

[0571] In the context of adoptive cell therapy, the administration of a given "dose" includes the administration of a given amount or quantity of cells as a single composition and / or a single uninterrupted administration (e.g., as a single injection or continuous infusion), and also includes the administration of a given amount or quantity of cells provided with multiple individual compositions or infusions as a fractionated dose over a specified time period (no more than 3 days). Therefore, in some cases, the first or continuous dose is a single or continuous administration of a specified number of cells given or started at a single time point. However, in some cases, the first or continuous dose is administered with multiple injections or infusions over a period of no more than three days, such as three or two days, once a day, or multiple infusions over a single day.

[0572] 3. Targeted degradation of pathogenic proteins

[0573] On the other hand, the present disclosure also provides a method for degrading a target protein in vivo or in vitro, which comprises delivering a nucleic acid molecule and / or a vector as described herein to a cell or subject expressing the target protein, wherein the nucleic acid molecule and / or the vector are expressed in the cell or subject to produce a chimeric protein construct as described herein. The targeting protein binding domain of the chimeric protein construct is capable of binding to the target protein, and the ERAD mechanism protein binding domain of the chimeric protein construct is capable of utilizing the ERAD pathway to induce spatial proximity of the ubiquitin-proteasome system (UPS), thereby utilizing the proteasome in the UPS to degrade the target protein. When the chimeric protein construct further comprises a protein degradation pathway member binding domain, the method will induce additional degradation systems to approach the target protein, so that the degradation effect is significantly improved.

[0574] On the other hand, the present disclosure also provides a method for treating a condition or disease in a subject in need thereof and / or preventing its recurrence, comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition described above (comprising, for example, the nucleic acid molecules described herein, such as mRNA, viral vectors, oncolytic viruses, chimeric protein constructs, etc.).

[0575] In certain embodiments, the method comprises 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.

[0576] Any conventional route of administration can be used in the context of the present invention, including parenteral, local or mucosal routes. Parenteral routes are injection or infusion administration, including systemic and local routes. Conventional parenteral injection types are intravenous (entering a vein), intraarterial (entering an artery), intradermal (entering the dermis), subcutaneous (below the epidermis), intramuscular (entering a muscle) and intratumoral (entering a tumor or a site very close to a tumor). Typical infusions are by intravenous route. Mucosal administration includes but is not limited to oral / esophageal, intranasal, tracheal, intrapulmonary, intravaginal, or intrarectal routes. Topical administration can also be accomplished by transdermal means (e.g., patches, etc. and so on). Conventional syringes and needles can be used to administer or any prior art compound or device that can promote or improve the delivery of an active agent in an object.

[0577] Depending on the disease that the individual needs to treat and the pathogenic protein that needs to be degraded, an appropriate chimeric protein construct, and its nucleic acid molecule and vector can be selected.

[0578] In some preferred embodiments, the diseases include various solid tumors and blood tumors, infectious diseases (such as viral infections), autoimmune diseases, neurodegenerative diseases, and metabolic diseases.

[0579] In some embodiments, the disease is a tumor or cancer, and the targeting domain of the chimeric protein construct comprises a target that specifically targets a tumor or an immune function. The tumor or immune function-related target is as described above in this application.

[0580] In some preferred embodiments, the solid tumor is selected from the group consisting of nervous system tumors, head and neck tumors, chest tumors, digestive system tumors, genitourinary system tumors, soft tissue and skin tumors, bone tumors, and the like.

[0581] In some preferred embodiments, nervous system tumors include diffuse gliomas, diffuse astrocytomas and anaplastic astrocytomas, glioblastomas, oligodendrogliomas, oligoastrocytomas, childhood diffuse gliomas, other astrocytomas, ependymomas, neuronal and mixed neuronal-glial tumors, medulloblastomas, other embryonal tumors, schwannomas, meningiomas, solitary fibrous tumors and hemangiopericytomas, etc.

[0582] In some preferred embodiments, head and neck tumors include nasal cavity and paranasal sinus malignancies, nasopharyngeal cancer, oral cancer, laryngeal cancer, salivary gland tumors, intracranial tumors, thyroid cancer, tongue cancer, etc.

[0583] In some preferred embodiments, the thoracic tumors include lung cancer, esophageal cancer, cardiac cancer, breast cancer, mediastinal tumors, and the like.

[0584] In some preferred embodiments, digestive system tumors include gastric cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, small intestinal malignancies, etc.

[0585] In some preferred embodiments, the genitourinary system tumors include renal cancer, prostate cancer, bladder cancer, testicular malignancies, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, and the like.

[0586] In some preferred embodiments, soft tissue and skin tumors include malignant fibrous histiocytoma, rhabdomyosarcoma, synovial sarcoma, malignant melanoma of the skin, and the like.

[0587] In some preferred embodiments, bone tumors include osteosarcoma, Ewing's sarcoma, and the like.

[0588] In some preferred embodiments, the colon cancer is a colon adenoma.

[0589] In some preferred embodiments, the breast cancer is triple-negative breast cancer cells.

[0590] In some preferred embodiments, the liver cancer is hepatocellular carcinoma.

[0591] In some preferred embodiments, the disease is a blood tumor selected from leukemia, lymphoma (HL), multiple myeloma (MM), myelodysplastic syndrome (MDS), etc.

[0592] In some preferred embodiments, the leukemia is B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute myeloid leukemia, etc.

[0593] On the other hand, the present disclosure also provides a method for treating a tumor or cancer in a subject in need thereof, comprising: administering a therapeutically effective amount of an oncolytic virus or an oncolytic virus pharmaceutical composition described herein to the subject. The oncolytic virus or composition of the present invention can be administered to the subject in a single dose or multiple doses. If multiple doses are administered, it can be administered by the same or different routes and can be administered at the same site or at different sites. Continuous circulation can also be used, repeated after a rest period. The interval between each administration can be from a few hours to a year (e.g., 24h, 48h, 72h, weekly, biweekly, monthly, or annually). The interval can also be irregular (e.g., after tumor development). Each administration dose can vary within the above range. In certain embodiments, the path for administering the oncolytic virus can include intravenous and intratumoral paths.

[0594] In the case of the present invention, a suitable dose (e.g. 10 7 to 5x10 9pfu) is administered with one or more oncolytic viruses. The interval between each viral administration can vary from about 1 day to about 8 weeks. Another preferred treatment regimen involves 2 to 5 (e.g., 3) intravenous or intratumoral administrations of 108 or 109 Pfu of oncolytic vaccinia virus, approximately 1 or 2 weeks apart, which can be selected by a doctor according to actual conditions. In some embodiments, the method includes administering an oncolytic viral vector comprising a nucleic acid molecule as described above to the subject, wherein the subject suffers from cancer. The pharmaceutical composition or the oncolytic viral vector comprising a nucleic acid molecule as described above can target and degrade pathogenic proteins, e.g., oncoproteins, viral proteins, immune-related proteins, etc.

[0595] In some embodiments, the disease is an infectious disease, and the targeting domain of the chimeric protein construct comprises a target site specifically targeting an infectious disease. The target site associated with the infectious disease is as described above in this application.

[0596] In some preferred embodiments, viral infectious diseases include: 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, slow virus infectious diseases, etc.

[0597] In some preferred embodiments, respiratory viral diseases include infections caused by rhinovirus, adenovirus, respiratory syncytial virus, parainfluenza virus, and coronavirus; influenza; mumps; and the like.

[0598] In some preferred embodiments, gastrointestinal viral diseases include poliomyelitis; Kuksaki virus infection; ECHO virus infection; viral gastroenteritis: including rotavirus gastroenteritis, Norwalk virus gastroenteritis, adenovirus gastroenteritis, astrovirus gastroenteritis, coronavirus gastroenteritis and calicivirus gastroenteritis, etc.

[0599] In some preferred embodiments, viral liver diseases include hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, Epstein-Barr virus hepatitis, cytomegalovirus hepatitis, and the like.

[0600] In some preferred embodiments, viral diseases of the skin and mucous membranes include measles, rubella, roseola infantum, varicella and herpes zoster, smallpox, herpes simplex virus infection, rabies and foot-and-mouth disease.

[0601] In some preferred embodiments, the ocular viral diseases include epidemic keratoconjunctivitis, follicular conjunctivitis, and herpes keratoconjunctivitis.

[0602] In some preferred embodiments, viral diseases of the central nervous system include Japanese encephalitis, Western equine encephalitis, Eastern equine encephalitis, St. Louis encephalitis, Venezuelan equine encephalitis, Murray Valley encephalitis, California encephalitis, forest encephalitis, and lymphocytic choriomeningitis.

[0603] In some preferred embodiments, the lymphocytic viral diseases include infectious mononucleosis, cytomegalovirus infection, and acquired immunodeficiency syndrome.

[0604] In some preferred embodiments, the insect-borne viral diseases include viral hemorrhagic fevers, including epidemic hemorrhagic fever, yellow fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever, Omsk hemorrhagic fever, Marburg disease and Ebola hemorrhagic fever; dengue fever and dengue hemorrhagic fever; West Nile fever; Colorado tick fever; sandfly fever, etc.

[0605] Preferably, the lentiviral infectious diseases include subacute sclerosing panencephalitis, kuru, progressive multifocal leukoencephalopathy and subacute spongiform encephalopathy (corticostriatal degeneration), etc.

[0606] In some embodiments, the disease is an autoimmune disease, and the targeting domain of the chimeric protein construct comprises a target specifically targeting an autoantigen-related target, as described above in this application.

[0607] In some preferred embodiments, autoimmune diseases include organ-specific autoimmune diseases and systemic autoimmune diseases.

[0608] Preferably, the organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, etc.

[0609] In some preferred embodiments, the systemic autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, etc.

[0610] In some embodiments, the disease is a neurological or degenerative disease, and the targeting domain of the chimeric protein construct comprises a target specifically targeting a neurological disease-related target as described above in this application.

[0611] In some embodiments, the neurological diseases include peripheral nerve diseases of the nervous system such as trigeminal neuralgia, facial palsy, hemifacial spasm, vestibular neuronitis, glossopharyngeal neuralgia, mononeuropathy, brachial plexus neuralgia, multiple mononeuropathy, polyneuropathy, acute inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy;

[0612] Spinal cord diseases such as myelitis, compressive myelopathy, subacute combined degeneration of the spinal cord, syringomyelia, spinal cord vascular disease, spinal arachnoiditis, etc.;

[0613] Cerebrovascular diseases such as transient ischemic attack, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, intracranial venous thrombosis, etc.;

[0614] Central nervous system infectious diseases such as meningitis, encephalitis caused by viral, bacterial, fungal or parasitic infections, and lentiviral encephalitis caused by lentiviral infections;

[0615] Demyelinating diseases of the central nervous system such as multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, leukodystrophy, etc.

[0616] Movement disorders such as Parkinson's disease, chorea, hepatolenticular degeneration, dystonia, essential tremor, tardive dyskinesia, etc.

[0617] epilepsy;

[0618] Headaches such as migraines, tension headaches, cluster headaches, etc.

[0619] Neurodegenerative diseases such as motor neuron disease, Alzheimer's disease, Lewy body dementia, frontotemporal dementia, multiple system atrophy, etc.

[0620] Genetic diseases of the nervous system such as hereditary ataxia, hereditary spastic paraplegia, Charcot-Marie-Tooth disease, neurofibromatosis, tuberous sclerosis, and cerebrofacial angiomatosis;

[0621] Neurological developmental disorders such as congenital hydrocephalus, cerebral palsy, basilar invagination, and cerebellar tonsillar disease;

[0622] Neuromuscular junction and muscle diseases such as myasthenia gravis, periodic paralysis, polymyositis, progressive muscular dystrophy, myotonic myopathy (myotonic dystrophy, myotonia congenita), metabolic myopathy (mitochondrial myopathy, lipid storage myopathy, glycogen storage disease), etc.

[0623] Autonomic nervous system diseases such as Raynaud's disease, erythromelalgia, hemifacial atrophy, systemic autonomic insufficiency, spontaneous hyperhidrosis, progressive lipodystrophy, etc.

[0624] Nervous system tumors such as glioma, lymphoma, meningioma, etc.:

[0625] Paraneoplastic syndromes of the nervous system such as paraneoplastic cerebellar degeneration, paraneoplastic encephalomyelitis, subacute necrotizing myelopathy, subacute motor neuron disease, paraneoplastic sensory neuron disease, etc.

[0626] In some preferred embodiments, neurodegenerative diseases result in a gradual loss of neuronal structure and function, including neuronal death and glial cell homeostasis, leading to cognitive impairments 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).

[0627] In some embodiments, the disease is a metabolic disease, such as diabetes, hyperlipidemia, gout, etc. The metabolic disease-related targets are as described above in this application.

[0628] Example

[0629] Example 1: Design of ER-TPD chimeric protein construct

[0630] This embodiment designs five structures of chimeric protein constructs based on the endoplasmic reticulum-based targeted protein degradation technology (ER-TPD). Among them, the first structure is a basic ER-TPD chimeric protein construct, and the other four structures are ER-TPD chimeric protein constructs with more powerful functions formed by further adding E3 ubiquitin ligase ligands, E2 ubiquitin-binding enzyme ligands or lysosomal ligands or combinations thereof on the basis of the first structure. The ER-TPD chimeric protein constructs contain a multifunctional integrated domain or functional variant of a recombinant protein molecule that binds to the target protein and causes its targeted degradation.

[0631] 1.1 Basic ER-TPD chimeric protein construct

[0632] In some embodiments, a basic ER-TPD chimeric protein construct TBD-TMD-ERD is used, whose linear schematic diagram and structural schematic diagram are shown in Figure 1A, which comprises three domains: (1) a targeting domain (TBD) or a functional variant thereof, which can be a targeted affinity ligand protein molecule or a functional variant thereof (which can be an antibody fragment, a polypeptide, a natural receptor, a ligand, or an artificial recombinant protein with target protein affinity or its binding domain); (2) a transmembrane domain (TMD) of a viral endoplasmic reticulum (ER) resident glycoprotein or a functional variant thereof; and (3) a cytoplasmic domain (ER retention domain, ERD) of a viral endoplasmic reticulum resident glycoprotein or a functional variant thereof. Optionally, the targeting domain or its functional variant, the transmembrane domain of a viral endoplasmic reticulum resident glycoprotein or its functional variant, and the cytoplasmic domain of a viral endoplasmic reticulum resident glycoprotein or its functional variant are connected by a hinge or a linker.

[0633] As shown in Figure 1B, the working structure 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, which causes the target protein to be retained in the endoplasmic reticulum, preventing the synthesized target protein from being transported to the Golgi apparatus. [2] Reverse transport: 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 through the reverse transporter in the ERAD mechanism. Classic reverse transporters include Derlin, the E3 ligase Hrd1, and its partner protein Sel1L, and can recruit the cytoplasmic ATPase p97. Derlin has six transmembrane domains, of which the N-terminus and C-terminus are both in the cytoplasm, forming a transport channel. At the same time, the reverse transport process requires the participation of ATPase p97 (Byun H et al. ERAD and how viruses exploit it. Front Microbiol. 2014 Jul 3; 5:330). [3] Ubiquitination and proteasomal degradation: The reverse transporter can interact with the endoplasmic reticulum's inherent E3 ubiquitin ligase to promote the ubiquitination of target proteins. The reverse transporter transports the ubiquitinated target proteins into the cytoplasm, where they are recognized and degraded by the proteasome.

[0634] 1.2 ER-TPD chimeric protein constructs with E3 ubiquitin ligase ligand domain

[0635] In some embodiments, an ER-TPD chimeric protein construct TBD-TMD-ERD-E3L having an E3 ubiquitin ligase ligand domain is used, and its linear schematic diagram and structural schematic diagram are shown in Figures 2A and 2B, respectively, which comprises four domains: (1) a targeting domain or a functional variant thereof, which can be a targeted affinity ligand protein molecule or a functional variant thereof (which can be an antibody fragment, a polypeptide, a natural receptor, a ligand, or an artificial recombinant protein domain with affinity for the target protein); (2) a transmembrane domain of a viral endoplasmic reticulum-resident glycoprotein or a functional variant thereof; (3) a cytoplasmic domain of a viral endoplasmic reticulum-resident glycoprotein or a functional variant thereof; and (4) an E3 ubiquitin ligase ligand (ligand for E3ligase, E3L) or a functional variant thereof, which can be a polypeptide or a recombinant protein used to connect to a large number of E3 ubiquitin ligases outside the endoplasmic reticulum. Optionally, the targeting domain or its functional variant, the transmembrane domain of the viral endoplasmic reticulum-resident glycoprotein or its functional variant, the cytoplasmic domain of the viral endoplasmic reticulum-resident glycoprotein or its functional variant, and the E3 ubiquitin ligase ligand domain or its functional variant are connected by a hinge or a linker. Due to the limited number of E3 ubiquitin ligases inherent in the endoplasmic reticulum, and also because the ERAD pathway of misfolded proteins in the endoplasmic reticulum competes for E3 ubiquitin ligases, efficient degradation of the target protein cannot be achieved without increasing the ubiquitination proteasome degradation pathway outside the endoplasmic reticulum. Based on this, the inventors of the present application have found that by exogenously increasing the E3 ubiquitin ligase ligand, efficient ubiquitination and degradation of the target protein can be achieved.

[0636] As shown in Figure 2C, the working structure 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, which causes the target protein to be retained in the endoplasmic reticulum, preventing the synthesized target protein from being transported to the Golgi apparatus. [2] Reverse transport: 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 through the reverse transporter in the ERAD mechanism. The classic reverse transporter includes Derlin, the E3 ligase Hrd1, and its partner protein Sel1L, and can recruit the ATPase p97 in the cytoplasm. Derlin has six transmembrane domains, of which the N-terminus and C-terminus are both in the cytoplasm, forming a transport channel. At the same time, the reverse transport process requires the participation of ATPase p97. [3] Ubiquitination and proteasomal degradation: The E3 ligase ligand in the chimeric protein construct binds to the E3 ligase complex and ubiquitin is ubiquitinated by the E2 ubiquitin-conjugating enzyme in the complex and transported to the proteasome for degradation. The ligand targeting the E3 ligase complex can be a protein sequence screened and constructed by human researchers, or a naturally occurring protein sequence or a recombinant sequence thereof, such as a phosphopeptide derived from IκBα (DRHDSGLDSM, SEQ ID NO. 119), which can recruit SCFβ-TRCP in the E3 ubiquitin ligase complex to mediate 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 USA. 2001; 98: 8554-9). It can also include an E3 ligase recognition peptide derived from HIF-1α (ALAPYIP, SEQ ID NO. 120), which is linked to the E3 ligase VHL through this recognition peptide, thereby achieving 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).

[0637] 1.3 ER-TPD chimeric protein constructs with E2 ubiquitin-binding enzyme ligand domain

[0638] In some embodiments, an ER-TPD chimeric protein construct TBD-TMD-ERD-E2L having an E2 ubiquitin-binding enzyme ligand domain is used, and its linear schematic diagram and structural schematic diagram are shown in Figures 3A and 3B, respectively, which comprises four domains: (1) a targeting domain or a functional variant thereof, which can be a targeted affinity ligand protein molecule or a functional variant thereof (which can be an antibody fragment, a polypeptide, a natural receptor, a ligand, or an artificial recombinant protein domain with affinity for the target protein); (2) a transmembrane domain of a viral endoplasmic reticulum-resident glycoprotein or a functional variant thereof; (3) a cytoplasmic domain of a viral endoplasmic reticulum-resident glycoprotein or a functional variant thereof; and (4) an E2 ubiquitin-binding enzyme ligand domain or a functional variant thereof, wherein the E3 ubiquitin-binding enzyme ligand can be a polypeptide or a recombinant protein. Optionally, the targeting domain or its functional variant, the transmembrane domain of the viral endoplasmic reticulum-resident glycoprotein or its functional variant, the cytoplasmic domain of the viral endoplasmic reticulum-resident glycoprotein or its functional variant, and the E2 ubiquitin-binding enzyme ligand domain or its functional variant are connected by a hinge or a linker.

[0639] As shown in Figure 3C, the working structure 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, which causes the target protein to be retained in the endoplasmic reticulum, preventing the synthesized target protein from being transported to the Golgi apparatus. [2] Reverse transport: 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 through the reverse transporter in the ERAD mechanism. The classic reverse transporter includes Derlin, the E3 ligase Hrd1, and its partner protein Sel1L, and can recruit the ATPase p97 in the cytoplasm. Derlin has six transmembrane domains, of which the N-terminus and C-terminus are both in the cytoplasm, forming a transport channel. At the same time, the reverse transport process requires the participation of ATPase p97. [3] Ubiquitination and proteasomal degradation: The chimeric protein construct directly binds to the E2 ubiquitin conjugating enzyme through the E2 ubiquitin conjugating enzyme ligand domain, and achieves ubiquitination of the target protein by the E2 ubiquitin conjugating enzyme and transports it to the proteasome for degradation. The E2 ubiquitin conjugating enzyme ligand domain can bind to the UBC structure of the E2 ubiquitin conjugating enzyme through the RING zinc finger domain derived from the IAP family protein, thereby achieving ubiquitination and proteasomal degradation of the target protein. The ligand targeting the E2 ubiquitin conjugating enzyme can be a RING zinc finger domain derived from the IAP family protein or a recombinant protein sequence with a RING zinc finger domain. IAPs (inhibitor of apoptosis proteins) are a family of inhibitory apoptosis proteins. They act as E3 ligases for pro-apoptotic proteins (such as caspases, SMAC, and ARTS), enabling their ubiquitination and degradation, thereby inhibiting apoptosis. They also regulate NOD signaling and immune responses by ubiquitinating RIPK2 (receptor-interacting serine / threonine-protein kinase 2). The five IAP family members (XIAP, cIAP1, cIAP2, Livin, and ILP2) all possess a RING zinc finger domain that binds to ubiquitin-binding enzymes (E2s) 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, the RING zinc finger domain derived from IAP family members can be used to link chimeric protein constructs and ubiquitin-conjugating enzymes E2, achieving more efficient target protein ubiquitination and proteasome-mediated degradation.The following sequence can be used as the sequence for designing E2 ligands:.

[0640] RING domain from XIAP: CKICMDRNIAIVFVPCGHLVTCKQCAEAVDKCPMCY (SEQ ID NO. 155);

[0641] RING domain from cIAP1: CKVCMDKEVSVVFIPCGHLVVCQECAPSLRKCPICR (SEQ ID NO. 156);

[0642] RING domain from cIAP2: CKVCMDKEVSVVFIPCGHLVVCQECAPSLRKCPICR (SEQ ID NO. 157);

[0643] RING domain from Livin: CKVCLDRAVSIVFVPCGHLVCAECAPGLQLCPICR (SEQ ID NO. 158);

[0644] RING domain from ILP-2: CKICMDRHIAVVFIPCGHLVTCKQCAEAVDRCPMCS (EQ ID NO. 159).

[0645] 1.4 ER-TPD chimeric protein constructs with lysosomal ligand domains

[0646] In some embodiments, an ER-TPD chimeric protein construct TBD-TMD-ERD-LL with a lysosomal ligand domain is used, whose linear schematic diagram and structural schematic diagram are shown in Figures 4A and 4B, respectively, and which comprises four domains: (1) a targeting domain or a functional variant thereof, which can be a targeted affinity ligand protein molecule or a functional variant thereof (which can be an antibody fragment, a polypeptide, a natural receptor, a ligand, or an artificial recombinant protein domain with affinity for the target protein); (2) a transmembrane domain of a viral endoplasmic reticulum resident glycoprotein or a functional variant thereof; (3) a cytoplasmic domain of a viral endoplasmic reticulum resident glycoprotein or a functional variant thereof; and (4) a lysosomal ligand domain or a functional variant thereof, which can be a polypeptide or a recombinant protein. Optionally, the targeting domain or a functional variant thereof, the transmembrane domain of a viral endoplasmic reticulum resident glycoprotein or a functional variant thereof, the cytoplasmic domain of a viral endoplasmic reticulum resident glycoprotein or a functional variant thereof, and the lysosomal ligand domain or a functional variant thereof are connected by a hinge or a linker.

[0647] As shown in Figure 4C, the working structural principle 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 on the inner membrane of the endoplasmic reticulum and binds to the target protein through the targeted target protein ligand, retaining it in the endoplasmic reticulum and preventing the synthesized target protein from being transported to the Golgi apparatus. [2] Reverse transport: 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 through the reverse transporter in the ERAD mechanism. [3] Lysosomal endocytosis and hydrolysis: The chimeric protein construct directly binds to the lysosomal molecular chaperone through the lysosomal ligand domain and is transported to the lysosome through the molecular chaperone. In the lysosome, various hydrolases degrade the target protein.

[0648] The lysosomal ligand domain can be a KFERQ-like motif or a recombinant protein sequence with a KFERQ-like motif. The KFERQ-like motif can be specifically recognized and bound by the molecular chaperone Hsc70, and through the molecular chaperone mediated autophagy (CMA) mechanism, the target protein ER-TPD complex is transported to the lysosome and degraded by various hydrolases.

[0649] Chaperone-mediated autophagy (CMA) is a selective protein autophagy and lysosomal degradation process. The molecular chaperone Hsc70 recognizes and targets KFERQ-like motifs on target proteins and, through the receptor protein LAMP2A on the lysosomal membrane, transports target proteins to lysosomes for degradation. The KFERQ-like motif may include 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 Annexin VI; RKVEQ (SEQ ID NO. 134) from aspartate aminotransferase; NLLKE (SEQ ID NO. 135) from c-fos; NRVVD (SEQ ID NO. 136) from GAPDH; NKKFE (SEQ ID NO. 137) from glutathione transferase; VKELQ (SEQ ID NO. 138) from IκB. NO.137); VDKLN derived from α-2-microglobulin (SEQ ID NO.138) and RIKEN (SEQ ID NO. 139); 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 the 26S proteasome (C8); QEKVF (SEQ ID NO. 144) from the 19S proteasome (PA28); QDLKF (SEQ ID NO. 145) from pyruvate kinase; and 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).

[0650] 1.5 ER-TPD chimeric protein constructs with E3 ubiquitination ligase and lysosomal ligand domains

[0651] In some embodiments, an ER-TPD chimeric protein construct TBD-TMD-ERD-E3L-LL having a combination of an E3 ubiquitin ligase ligand and a lysosomal ligand is used, the linear schematic diagram and structural schematic diagram of which are shown in Figures 5A and 5B, respectively, and which comprises five domains: (1) a targeting domain or a functional variant thereof, which can be a targeted 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 with affinity for the target protein); (2) a transmembrane domain of a viral endoplasmic reticulum-resident glycoprotein or a functional variant thereof; (3) a cytoplasmic domain of a viral endoplasmic reticulum-resident glycoprotein or a functional variant thereof; (4) an E3 ubiquitin ligase ligand or a functional variant thereof (ligand for E3ligase, E3L) and (5) a lysosomal ligand or a functional variant thereof (Ligand for lysosome). Optionally, the targeting domain or its functional variant, the transmembrane domain of the viral endoplasmic reticulum-resident glycoprotein or its functional variant, the cytoplasmic domain of the viral endoplasmic reticulum-resident glycoprotein or its functional variant, the E3 ubiquitin ligase ligand or its functional variant, and the lysosomal ligand domain or its functional variant are connected by a hinge or a linker.

[0652] As shown in Figure 5C, the working structure 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, which causes the target protein to be retained in the endoplasmic reticulum and prevents the synthesized target protein from being transported to the Golgi apparatus. [2] Reverse transport: 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 through the reverse transporter in the ERAD mechanism. [3] Lysosomal endocytosis and enzymatic degradation as well as ubiquitination / proteasome degradation: The chimeric protein construct is transported outside the endoplasmic reticulum, directly binds to the lysosomal molecular chaperone through the lysosomal ligand domain, and is transported to the lysosome through the molecular chaperone. In the lysosome, various hydrolases degrade the target protein; at the same time, some chimeric proteins are transported outside the endoplasmic reticulum: the E3 ligase ligand in the chimeric protein construct can bind to the E3 ligase complex, and ubiquitinate the target protein through the E2 ubiquitin-binding enzyme in t...

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