Targeted protein degradation systems and their applications

Abstract

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

Description

[Technical Field]

[0001] This disclosure relates to targeted protein degradation systems and their applications, particularly to applications in disease treatment (including, but not limited to, cancer, viral infections, autoimmune diseases, and neurodegenerative diseases). [Background technology]

[0002] Targeted removal of proteins is necessary for at least the following reasons: 1) determining the function of a particular protein in basic research; 2) validating therapeutic targets; 3) inhibiting the action of pathogenic proteins (e.g., tumor proteins or proteins that cause neurodegeneration); 4) accelerating the degradation of proteins that accumulate during the aging process; and 5) mitigating side effects of cell therapy (e.g., inhibiting the expression of immunogenic proteins). However, conventional techniques for targeted removal of proteins each have their own unique technical problems. Gene editing technologies (such as CRISPR technology) can target and knock out genes encoding arbitrary target proteins at the gene level, but they have the problem of off-target effects, low specificity in recognizing and cleaving desired DNA sites, and are prone to side effects such as chromosomal instability.

[0003] Small interfering RNAs (siRNAs) can inhibit the synthesis of target proteins at the RNA level, but siRNAs also have potential off-target effects. For example, the sense strand of siRNA can mediate the expression silencing of congeneral genes, causing sense-strand-mediated off-target effects. Furthermore, small mismatches in small nucleic acid sequences can also cause silencing of other genes, leading to off-target effects and potentially toxic or side effects. Proteolysis-targeting chimera (PROTAC) technology can degrade target proteins at the protein level, and is usually added to cells as a biochemical reagent or administered to animals or humans to induce the degradation of target proteins in cells. Early PROTACs were based on E3 ligase-binding polypeptide ligand molecules, but PROTACs prepared using polypeptide ligand molecules have low cell permeability and are unstable, resulting in low degradation efficiency. PROTACs using small molecules as E3 ligase ligands have shown remarkable effectiveness, but their membrane permeability and bioavailability are poor. Accordingly, there is an urgent need for a novel target protein degradation system to overcome at least one problem existing in various conventional techniques for targeted removal of target proteins. Summary of the Invention Problems to be Solved by the Invention

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

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

[0006] Embodiment 6. A chimeric protein construct according to any one of Embodiments 1 to 5, further comprising a protein degradation pathway member (e.g., E3 ubiquitin ligase, proteasome, lysosome) binding domain which may be linked to the ERAD mechanism protein binding domain. Embodiment 7. A chimeric protein construct according to any one of Embodiments 1 to 6, wherein the targeting domain comprises an antibody or a functional fragment thereof that specifically targets a target protein (e.g., Fd, Fv, Fab, Fab', F(ab')2, Fv(scFv), single-chain antibody (scFv), nanobody, double-chain antibody, triple-chain antibody, and quadruple-chain antibody). Embodiment 8. The chimeric protein construct according to Embodiment 7, wherein the target protein is a pathogenic protein, and the pathogenic protein may be 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. Embodiment 9. The autoantigen proteins include type 1 diabetes-related autoantigens such as pancreatic islet cell antigen (ICA), insulin (IAA), glutamate decarboxylase 65 (GAD65), and insulinoma antigen-2 (IA-2); citrullinated protein / peptide antibodies, heterogeneous nuclear ribonucleoprotein A2 / B1, aldolase, alpha-enolase, calreticulin, heat shock protein (HSP60), BiP, PGK1, stress-induced phosphoprotein 1, and rheumatoid arthritis (RA)-related autoantigens such as FUSE-BP1 / 2; deoxyribonucleoprotein, SmD1, SmD3, Clq, lupus anticoagulant (LA), cardiolipin (CL), and β2 glycoprotein I (β2 GP). I) Systemic lupus erythematosus (SLE)-related autoantigens such as prothrombin (PT) and phosphatidylserine (PS); systemic sclerosis (SSc) / scleroderma (SD)-related autoantigens such as Scl-70, SSA, and Ro52; autoimmune liver disease-related antigens such as mitochondrial antigens, Spl00, PML, gp210, and p62; myasthenia gravis-related autoantigens such as acetylcholine receptors; central nervous system receptors 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, and glycine receptor. Autoantigens associated with autoimmune diseases of the nervous system (limbic encephalitis, encephalomyelitis, cerebellar ataxia); 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-induced irritable bowel syndrome, 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);A chimeric protein construct according to Embodiment 8, selected from the group consisting of reproductively related autoantigens such as ovarian antigens and sperm antigens.

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

[0008] Embodiment 11. The pathogenic proteins include HBV surface antigen, HBV capsid glycoprotein, HBeAg, HBV DNA polymerase, HBV code X protein (HBx), HIV Gag protein, HIV Env protein, HIV gp120, HIV-1 reverse transcriptase, HIV gp120, HCV NS3-4A protease, HCV RNA polymerase, HCV envelope protein, EBV DNA polymerase, EBV EBNA1, coronavirus RNA synthetase, coronavirus spike protein, coronavirus envelope protein, coronavirus membrane protein, coronavirus nucleocapsid protein, RNA-dependent RNA polymerase (RdRp) such as novel coronavirus RNA-dependent RNA polymerase, herpesviruses DNA / RNA polymerase, herpesvirus capsid glycoprotein, and CMV. A chimeric protein construct according to several embodiments 8, comprising a virus-associated protein selected from DNA polymerase, CMV capsid glycoprotein, RSV envelope protein, RSV capsid protein, RSV RNA polymerase, influenza virus RNA polymerase, influenza virus envelope protein, HPV DNA polymerase, and HPV capsid protein.

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

[0010] Embodiment 17. A chimeric protein construct according to several embodiments 16, wherein the viral endoplasmic reticulum commensal protein is at least one selected from the group consisting of HCMV glycoproteins US2, US11, US3, US10, US6, HSV ICP47, CPXV12, BHV UL49.5, EBV BNFL2a, HCMV UL16, UL141, UL142, HIV Nef, HIV Vpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3, HTLV-1 p12, and cowpox virus protein CPXV203. Embodiment 18. A chimeric protein construct according to any one of Embodiments 1 to 17, linked to at least one co-expression moiety. Embodiment 19. A chimeric protein construct according to Embodiment 18, wherein the at least one co-expression moiety is independently selected from intact viral ER commensal glycoproteins (e.g., HCMV US2, US3, US11, US10, adenovirus E3-Kl9, HCMV US6, HSV ICP47), chimeric antigen receptors (CARs), functional T cell receptors (TCRs), chemokine receptors, and NK cell activating receptors. Embodiment 20. A chimeric protein construct according to Embodiment 19, wherein the chemokine receptor is selected from CCR4, CCR5, CCR6, CCR7, CCR9, CCR2b, CXCR1, CXCR2, and CXCR4. Embodiment 21. A chimeric protein construct according to Embodiment 20, wherein the NK cell activating receptor comprises (a) the extracellular domain (ED) or a functional variant thereof of the NK cell activating receptor, (b) the transmembrane domain (TMD) or a functional variant thereof of the NK cell activating receptor, and (c) the intracellular domain (ICD) or a functional variant thereof of the NK cell activating receptor; and a hinge or linker may be included between the extracellular domain or a functional variant thereof of the NK cell activating receptor, the transmembrane domain or a functional variant thereof of the NK cell activating receptor and / or the intracellular domain or a functional variant thereof of the NK cell activating receptor. Embodiment 22. A chimeric protein construct according to Embodiment 21, wherein the NK cell activation receptor is selected from NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, native cytotoxic receptor, TRAIL, DNAM-1, CD16a, 2B4, NTB-A, CRACC, and NKp80.

[0011] Embodiment 23. The chimeric protein construct according to Embodiment 21 or 22, wherein the NK cell activating receptor forms a complex with a CNK signaling module. Embodiment 24. A chimeric protein construct according to any one of embodiments 18 to 23, wherein the at least one co-expression portion is linked to the chimeric protein construct by a cleavable linker. Embodiment 25. A nucleic acid molecule encoding a chimeric protein construct according to any one of Embodiments 1 to 24. Embodiment 26. Nucleic acid molecules according to some Embodiment 25, including deoxyribonucleic acid (DNA), ribonucleic acid (RNA) (e.g., mRNA, circular RNA, ccRNA), threose nucleic acid (TNA), ethylene glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (including LNA, LNA having a β-D-ribose configuration, α-LNA having an α-L-ribose configuration (diastereoisomers of LNA), 2'-amino-LNA having 2'-amino functionalization, and 2'-amino-α-LNA having 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), and / or chimeric forms and / or combinations thereof. Embodiment 27. A vector comprising a nucleic acid molecule as described in Embodiment 25 or 26, wherein the nucleic acid molecule is operably ligated to at least one polynucleotide regulator to express a chimeric protein construct encoded by the nucleic acid molecule. Embodiment 28. The vector according to Embodiment 27, wherein the vector is selected from plasmids, nanoplasmides, cosmids, viral vectors (e.g., oncolytic viral vectors), minicircles, RNA vectors, or linear or round DNA (e.g., transposon DNA) or RNA molecules.

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

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

[0014] Embodiment 41. The method according to several embodiments 38, wherein the stem cells are hematopoietic progenitor cells (e.g., T cell progenitor cells, NK cell progenitor cells, macrophage progenitor cells), hematopoietic stem cells (HSCs), CD34+ cells, embryonic stem cells, mesenchymal stem cells, or iPSC cells. Embodiment 42. The method according to several embodiments 37, wherein the cells are immune cells. Embodiment 43. The method according to several embodiments 40, wherein the immune cells are T cells, natural killer (NK) cells, B cells, macrophages, monocytes, or dendritic cells. Embodiment 44. The method according to several embodiments 41, wherein the cells are T cells selected from a group of cells consisting of CD4+ T cells, CD8+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, natural killer T cells, γ-δ T cells, cytokine-induced killer (CIK) T cells, tumor-infiltrating lymphocytes, and dominant T cells. Embodiment 45. A cell population generated ex vivo by the method described in any one of Embodiments 39 to 44. Embodiment 46. (i) a chimeric protein construct according to any one of Embodiments 1 to 24 or a nucleic acid molecule according to Embodiment 25 or 26 or a vector according to any one of Embodiments 27 to 34 or an engineered cell according to any one of Embodiments 35 to 38 or a cell population according to Embodiment 45, and (ii) a pharmaceutical composition or kit comprising a pharmaceutically acceptable medium. Embodiment 47. A method for degrading a target protein, comprising delivering a vector according to any one of Embodiments 27 to 34 to a cell expressing the target protein. Embodiment 48. The method according to Embodiment 47, wherein the cells are selected from tumor cells, virus-infected cells, nerve cells, transplanted cells, and immune cells. Embodiment 49. The method according to Embodiment 47 or 48, wherein the delivery is by a viral vector (e.g., oncolytic virus). Embodiment 50. The method according to Embodiment 49, wherein the vector expresses the chimeric protein construct described in any one of Embodiments 1 to 24 in the cells. Embodiment 51. The method according to Embodiment 50, wherein the chimeric protein construct can simultaneously bind to a target protein and an ERAD mechanism protein, thereby degrading the target protein. Embodiment 52. A method for treating a condition or disease of a subject requiring treatment, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described in Embodiment 46.

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

[0016] [Figure 1]This shows chimeric protein constructs obtained by endoplasmic reticulum-based targeted protein degradation technology. Here, Figure 1A is a linear schematic and structural schematic of the basic ER-TPD chimeric protein construct TBD-TMD-ERD; Figure 1B shows the mechanism of action of the ER-TPD chimeric protein construct TBD-TMD-ERD. [Figure 2] The ER-TPD chimeric protein construct TBD-TMD-ERD-E3L, which has an E3 ubiquitin ligase ligand domain, is shown. Here, Figure 2A is its linear schematic diagram, Figure 2B is its structural schematic diagram, and Figure 2C shows the mechanism of action of the ER-TPD chimeric protein construct TBD-TMD-ERD-E3L. [Figure 3] The ER-TPD chimeric protein construct TBD-TMD-ERD-E2L, which has an E2 ubiquitous-conjugating enzyme ligand domain, is shown. Here, Figure 3A is its linear schematic diagram, Figure 3B is its structural schematic diagram, and Figure 3C shows the mechanism of action of the ER-TPD chimeric protein construct TBD-TMD-ERD-E3L. [Figure 4] The ER-TPD chimeric protein construct TBD-TMD-ERD-LL, which has a lysosomal ligand domain, is shown. Here, Figure 4A is its linear schematic diagram, Figure 4B is its structural schematic diagram, and Figure 4C shows the mechanism of action of the ER-TPD chimeric protein construct TBD-TMD-ERD-LL. [Figure 5] The ER-TPD chimeric protein construct TBD-TMD-ERD-E3L-LL, which has a combination of an E3 ubiquitinated ligase ligand and a lysosomal ligand, is shown. Here, Figure 5A is its linear schematic diagram, Figure 5B is its structural schematic diagram, and Figure 5C shows the mechanism of action of the ER-TPD chimeric protein construct TBD-TMD-ERD-E3L-LL. [Figure 6] The ER-TPD chimeric nucleic acid construct TCR-ER-TPD is shown. Here, Figure 6A shows the chimeric nucleic acid construct TCR-TPD1; Figure 6B shows the chimeric nucleic acid construct TCR-TPD2. [Figure 7] The results of detecting the cell phenotype of TCR ER-TPD Jurkat cells at 48 h (A), 96 h (B), and day 6 (C) are shown. [Figure 8] The following shows expression vectors designed with the EF1α promoter as the promoter using a single lentiviral expression vector. Here, Figure 8A shows the CNK expression vector; Figure 8B shows the chimeric protein construct HLA-ER-TPD; and Figure 8C shows the chimeric protein construct CNK-HLA-ER-TPD. [Figure 9] The flow cytometry results of cellular phenotypes 48 hours after transfecting 293 cells with both an ER-TPD chimeric nucleic acid construct targeting HLA-abc and a CNK expression vector are shown. [Figure 10] The ER-TPD chimeric nucleic acid construct that targets CD123 is shown. Here, Figure 10A shows the CNK expression vector; Figure 10B shows the chimeric protein construct HLA-TPD. [Figure 11] This shows the flow cytometry results of the cell phenotype 48 hours after transfecting the acute myeloid leukemia cell line THP1 with an scFv TPD chimeric nucleic acid construct targeting CD123. [Figure 12] This section describes the application of ER-TPD-CD123 CAR-T cells that target CD123. Here, Figure 12A1 shows the CD123 CAR, Figure 12B1 shows the expression of CD123 in activated T cells, and Figure 12C1 shows that CAR-T cells that target CD123 undergo a suicide effect in activated culture and tumor cell killing processes; Figure 12A2 shows the CD123 CAR-T-T2A-CD123 ER-TPD chimeric protein construct, Figure 12B2 shows that expressing this chimeric protein construct can degrade self-expressed CD123, and Figure 12C2 shows the tumor cell killing effect after expression of this chimeric protein construct. [Figure 13]This paper demonstrates the application of ER-TPD chimeric protein constructs that target autoantibodies in the treatment of autoimmune diseases. Here, Figure 13A shows that in plasma cells of type 1 diabetes patients, insulin antibodies are expressed and released, and these antibodies bind to insulin, removing insulin from the peripheral blood and significantly reducing the patient's insulin utilization rate; Figure 13B shows that ER-TPD transposon DNA that targets insulin antibodies is delivered into plasma cells using liposomes that target plasma cells, achieving integration and long-term expression of the IAA ER-TPD chimeric protein construct. [Figure 14] Exemplary chimeric fusion proteins containing one or more transmembrane and cytoplasmic domains of a TCRαβ or HLA molecule binding molecule and a viral ER commensal protein were described. Here, Figure 14A is a schematic diagram and composition of a UT chimeric multidomain. The broad structure of one viral ER commensal glycoprotein (VEP) consists of a luminal domain (LD), a transmembrane domain (TMD), and a cytoplasmic functional domain; the UT chimeric multidomain consists of a TCR or HLA binding domain, a hinge / spacer, a VEP transmembrane, and a cytoplasmic domain. Figure 14B is a schematic diagram and composition of a UT chimeric multidomain. The broad structures of different viral ER commensal glycoproteins (VEPs) consist of different luminal domains (LD), transmembrane domains (TMD), and cytoplasmic functional domains; the UT chimeric multidomain consists of a TCR or HLA binding domain, a hinge / spacer, and is different from the VEP transmembrane and cytoplasmic domains. Figure 14C is a schematic diagram and composition of a UT chimeric multistructure. The broad structures of different viral ER commensal glycoproteins (VEPs) consist of different luminal domains (LDs), transmembrane domains (TMDs), and cytoplasmic functional domains; UT chimeric multidomains consist of a TCR or HLA-binding domain, a hinge / spacer, and different VEP transmembrane domains and chimeric cytoplasmic domains are derived from different VEPs. [Figure 15]This study demonstrates that forced expression of the UT element can effectively inhibit TCR expression in Jurkat cells. A lentiviral vector containing cleaved EGFR was constructed, encoding anti-TCR scFv that fused with different viral ER commensal proteins, HCMV US2, US3, US11, and the adenovirus E19™ / CT domain, and anti-TCR scFv that fused with US2 and adenovirus E19™ / CT. This lentivirus was transduced into Jurkat cells, with untransduced Jurkat cells used as a control. 48 hours after transduction, cells were subjected to flow cytometry to detect TCRαβ expression in transduced cells (EGFR+ cells). The results showed that: all EGFR(+) Jurkat cells exhibited clear downregulation of TCRαβ expression; cells transduced in combination with the UT component (US2 / E19) could effectively inhibit TCRαβ expression. All tested UT chimeric elements (US2, US3, US11, E19) inhibited TCRαβ expression. Combining UT elements (US2 and E19) significantly enhanced the inhibitory effect on TCRαβ expression on the surface of Jurkat cells. [Figure 16]This study demonstrates that forced expression of the UT element can effectively inhibit TCR expression in human T cells. Figure 16A shows that forced expression of the UT element can effectively inhibit TCR expression in human T cells. A lentiviral vector containing a truncated EGFR expression cassette encodes anti-TCR scFv (anti-TCR-US2TM / CT-T2A-EGFRt) fused to the HCMV US3TM / CT domain. Human T cells were stimulated and transduced with this lentivirus, while untransduced T cells were used as a control. Five days after transduction, cells were subjected to flow cytometry to detect TCRαβ expression in transduced T cells (EGFR+ cells). The results showed that EGFR(+)T cells exhibited significant downregulation of TCRαβ expression compared to untransduced T cells. This demonstrates that the US element containing anti-TCR-US3TM / CT can effectively inhibit TCRαβ expression in human T cells. Figure 16B shows that forced expression of the UT element can effectively inhibit TCR expression in human T cells. A lentiviral vector with an expression cassette containing cleaved EGFR encodes anti-TCR scFv (anti-TCR-E19TM / CT-T2A-EGFRt) fused to the HCMV E19TM / CT domain. Human T cells were stimulated and transduced with this lentivirus, and untransduced T cells were used as a control. Five days after transduction, cells were subjected to flow cytometry to detect TCRαβ expression in transduced T cells (EGFR+ cells). The results showed that EGFR(+)T cells exhibited clear downregulation of TCRαβ expression compared to untransduced T cells. Increased viral transduction can further enhance the inhibition of TCRαβ expression on the T cell surface. This demonstrates that the US element containing anti-TCR-E19TM / CT can effectively express TCRαβ in human T cells. [Figure 17]CNK-T cells containing the UT element showed downregulation of TCR expression in CD8 / CD4 cells. The lentiviral vector contained an expression cassette (CNK-UT-E19) encoding a chimeric NK receptor and anti-TCR scFv fused to the E19TM / CT domain. This lentivirus was transduced into human T cells. Five days after transduction, the cells were subjected to flow cytometry to detect TCRαβ expression in transduced cells (NKG2D+ cells), T cells transduced with the conventional chimeric NK receptor, and untransduced T cells. The results showed the following: The introduction of the UT element into the CNK expression cassette does not affect the expression of the chimeric NK receptor. Furthermore, it can significantly inhibit TCR expression in CD8+ and CD4+ CNK-T cells. [Figure 18] This study demonstrates that CNK-T cells possessing the UT element exhibited strong cytotoxicity against tumor cells, similar to that of normal CNK-T cells. Non-transgenic T cells, CNK-T cells, and CNK-UT(E19) cells were co-cultured with HepG2 cells in an E:T ratio of 1:5. After 24 hours of culture, cells were collected and analyzed by flow cytometry to detect tumor lysis and T cell activation. The data revealed the following: CNK-UT cells exhibited strong cytotoxicity against HepG2 cells, comparable to that of normal CNK-T cells. After 24 hours of co-culture, CD45(-) tumor cells were significantly reduced in co-cultures of CNK-T and CNK-UT cells. Furthermore, after co-culturing CD8 and CD4 CNK-UT cells with HepG2 cells, both CD25 and CD137 were upregulated. [Figure 19]Figure 19A shows the basic CNK-UT multi-functional complex; Figure 19B shows the CNK-UT multi-functional complex with the addition of a binding protein molecular domain that targets MHC I; Figure 19C shows the CNK-UT multi-functional complex with the addition of a binding protein molecular domain that targets MHC I and a chimeric adapter; and Figure 19D shows the CNK-UT multi-functional complex with the addition of a binding protein molecular domain that targets MHC I and receptors that target and kill tumor cells such as CARs or TCRs. [Figure 20] The structures of the four CNK-UT elements expressing the CNK-UT multifunctional complex are shown. Here, as shown in Figure 20A, one combination of CNK-UT elements, DAP10-DAP12 ICD-T2A-NKG2D-p2A-anti-TCR-AdE3 ERAD, is initiated by a single lentiviral EF1α promoter expression vector; as shown in Figure 20B, one combination of CNK-UT elements, DAP10-DAP12 ICD-T2A-NKG2D-p2A-anti-TCR-US2 ERAD, is initiated by a single lentiviral EF1α promoter expression vector; and as shown in Figure 20C, DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TAA scFv-DAP10 and anti-TCR-AdE3 are initiated by the EF1α and CMV promoters, respectively, using a single lentiviral expression vector. We achieved the regulation of the expression of multiple ERAD-E2A-AdE3 genes and the expression of multiple functional elements with a single vector; as shown in Figure 20D, we regulated the expression of anti-TAA scFv-CD28 / 4-1BB-CD3ζ-T2A-CD3ζ-p2A-NKG2D and anti-TCR-AdE3 ERAD-T2A-AdE3, respectively, using two different lentiviral expression vectors, and transfected them together into T cells to achieve the expression of multiple CNK-UT functional elements in the same T cells. [Figure 21]The flow cytometry results for the basic phenotype of CNK-UT (DAP10-CD3ζ-T2A-NKG2D-p2A-anti-TCR scFv-AdE3) cells are shown. [Figure 22] The results of CNK-UT cell recognition and specific killing of the human colon adenoma cell line HT29 are shown. Here, Figure 22A shows the expression status of different NK target proteins in the human colon adenoma cell line HT29; Figure 22B shows that CNK-UT cells have effective killing and activating functions against HT29. [Figure 23] This demonstrates the recognition and specific killing of MDA-MB453 by CNK-UT cells. Here, Figure 23A shows the expression status of different NK target proteins in the triple-negative breast cancer cell line MDA-MB453; Figure 23B shows that CNK-UT cells exhibit effective killing and activating functions against THP1. [Figure 24] Figure 25 shows that GPC3 CAR / CNK-UT cells have a more effective tumor removal ability than GPC3 CAR-T cells in mice. [Figure 25] This study demonstrates that GPC3 CAR / CNK-UT cells have a more effective tumor removal capacity than GPC3 CAR-T cells. [Modes for carrying out the invention]

[0017] Before describing this disclosure in more detail, please understand that this disclosure is not limited to the specific embodiments described and is, of course, subject to change. Also, please understand that the scope of this disclosure is limited only by the appended claims, and that the terms used herein are intended solely to describe specific embodiments and are not intended to limit them. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this disclosure, but preferred methods and materials are described herein.

[0018] All publications and patents cited herein are incorporated herein by reference, to the extent that each individual publication or patent is specifically and individually indicated as being incorporated by reference, and are incorporated herein by reference to disclose and describe methods and / or materials relating to the cited publications. Any citation of a publication is for a disclosure prior to the filing date, and this disclosure should not be construed as acknowledging that such publication has no prior rights by prior disclosure. Furthermore, the publication dates provided may differ from the actual publication dates that need to be individually verified. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described herein has separate components and features that can be readily separated or combined with features of any of several other embodiments without departing from the scope or spirit of this disclosure. Any method described may be carried out in the order of the events described, or in any other logically possible order.

[0019] I. Definition The following definitions are provided for the assistance of the reader. Unless otherwise defined, all technical terms, symbols, and other scientific or medical terms or specialized terms used herein are intended to have meanings that are generally understood by those skilled in the art. In some cases, terms that have generally 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 representing a substantial difference from the definitions of terms that are generally understood in the art. In this disclosure, terms such as “comprises / comprised / comprising” and “contains / containing” have the meanings defined in U.S. patent law, and these are comprehensive or open, not excluding the addition of elements or method steps not described. The terms “consisting essentially of / consists essentially of” have the meanings defined in U.S. patent law, and these may include additional components or steps that do not substantially affect the basic and novel features of the invention to be protected. The terms “consists of / consisting of” have the meanings defined in U.S. patent law, meaning these terms are closed. As used herein, unless the context clearly indicates otherwise, the singular forms "a / an" and "the aforementioned" include multiple subjects.

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

[0021] As used herein, the terms “functional fragment” and “antigen-binding fragment” of an antibody may be used interchangeably and refer to a fragment of an antibody containing one or more CDRs, or an antibody fragment formed from any other antibody portion that binds to an antigen but does not contain the complete original antibody structure. Specific examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-bonded Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-bonded diabodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding fragment can bind to the same antigen to which the parent antibody binds. In some embodiments, an antigen-binding fragment may contain one or more CDRs derived from a particular parent antibody. The "Fab" of an antibody refers to a monovalent antigen-binding fragment, consisting of a single light chain (variable and constant regions) bound to the variable and first constant regions of a heavy chain via disulfide bonds. The Fab can be obtained by digesting the antibody with papain at the residues near the N-terminus of the disulfide bonds between the heavy chains in the hinge region. "Fab'" refers to a Fab fragment that includes part of the hinge region. It can be obtained by digesting the antibody with pepsin at residues near the C-terminus of the disulfide bond between the heavy chains of the hinge region. Therefore, a few residues in the hinge region (including one or more cysteines) differentiate it from Fab. "F(ab')2" refers to the dimer of Fab', which contains two light chains and parts of two heavy chains. The "Fv" in antibody terminology refers to the smallest antibody fragment that possesses a complete antigen-binding site. An Fv fragment consists of a variable region of one light chain bound to a variable region of one heavy chain. "dsFv" refers to a disulfide bond-stabilized Fv fragment, where the bond between the variable region of one light chain and the variable region of one heavy chain is a disulfide bond.

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

[0023] "Camelized single-domain antibody," "heavy-chain antibody," "nanobody," or "HCAb" refers to an antibody that contains two VH domains but does not contain a light chain (Riechmann L. and Muyldermans S., "J Immunol Methods". December 10;231(1-2):25-38 (1999); Muyldermans S., "J Biotechnol". June;74(4):277-302 (2001);WO94 / 04678;WO94 / 25591;US No. 6,005,079). Heavy-chain antibodies were originally obtained from the camelid family (Camelidae) (camels, dromedaries, and llamas). Camelized antibodies do not contain light chains but possess a true antigen-binding repertoire (Hamers-Casterman C. et al., "Nature". June 3; 363(6428):446-8 (1993); Nguyen VK. et al., "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation" "Immunogenetics". April; 54(1):39-47 (2002); Nguyen VK. et al., "Immunology". May; 109(1):93-101 (2003)). The variable domain (VHH domain) of heavy chain antibodies represents the smallest known antigen-binding unit generated from an adaptive immune response (Koch-Nolte F. et al., Journal of the Federation of American Societies for Experimental Biology (FASEB J.), November;21(13):3490-8. Electronic edition June 15, 2007 (2007)). A "diabody" contains a small antibody fragment having two antigen-binding sites, the fragment containing a VH domain (VH-VL or VL-VH) linked to a VL domain in a single polypeptide chain (see, for example, Holliger P. et al., Journal of the National Academy of Sciences, July 15;90(14):6444-8 (1993);EP404097;WO93 / 11161). Due to the short linker, the two domains in the same chain cannot be paired. Therefore, the aforementioned domain pairs with a complementary domain on another chain, generating two antigen-binding sites.The antigen-binding site can target the same or different antigens (or epitopes).

[0024] A "domain antibody" refers to an antibody fragment that contains only the variable region of the heavy chain or the variable region of the light chain. In some embodiments, two or more VH domains are covalently linked via a peptide linker to form a bivalent or polyvalent domain antibody. The two VH domains of a bivalent domain antibody can target the same or different antigens. In some embodiments, "(dsFv)2" comprises three peptide chains, with two VH portions linked via peptide linkers and two VL portions linked by disulfide crosslinks. In some embodiments, the “bispecificity ds diabody” includes VH1-VL2 (linked by a peptide linker) which is linked to VL1-VH2 (also linked by a peptide linker) via a disulfide crosslink between VH1 and VL1. In some embodiments, “bispecific dsFv” or “dsFv-dsFv'” comprises three peptide chains, where the heavy chain of the VH1-VH2 moiety is linked by a peptide linker (e.g., a long flexible linker) and pairs with the VL1 and VL2 moieties, respectively, via disulfide crosslinks. The heavy and light chains paired via a single disulfide bond have different antigen specificities. "Autologous cells" refer to any cells that originate from the same individual as the recipient of these cells (i.e., the recipient itself).

[0025] "Allogeneic cells" refer to any cells originating from another individual of the same species as the recipient of this cell (i.e., not the recipient itself). As used herein, the terms “construct” and “protein construct” may be used interchangeably and 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 is capable of performing functions that the individual proteins within it cannot perform. In the complex, the constituent proteins may or may not be in contact with each other. As used herein, the term "chimera," when relating to a structure, means that a part of the structure originates from one species and another part of the structure originates from another species.

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

[0027] As used herein, the phrase “host cell” means a cell into which an exogenous polynucleotide and / or vector has been introduced or into which it is to be introduced. As used herein, the term "functional variant" refers to a polypeptide / protein that has at least 70% sequence identity with the parent polypeptide / protein but still retains the function of the parent polypeptide / protein. The variant differs from the parent polypeptide / protein by one or more amino acid residues. For example, a functional variant may have one or more amino acid residues of the parent polypeptide / protein that have been substituted, added, deleted, inserted, or cleaved.

[0028] In this application, the percentage of "identity" or "sequence identity" of a polypeptide or polynucleotide is determined by comparing two sequences that are optimally aligned in a comparison window. For optimal alignment of the two sequences, portions of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) relative to the reference sequence (which does not contain additions or deletions). The percentage is calculated as follows: Determine the number of positions in which the same nucleic acid base or amino acid residue appears in the two sequences, find the number of matching positions, and divide the number of matching positions by the total number of positions in the comparison window and multiply the result by 100 to obtain the percentage of sequence identity. Alignment for the purpose of determining the sequence identity percentage of amino acids (or nucleic acids) can be performed using publicly available tools, such as BLASTN, BLASTp (available on the website of the National Center for Biotechnology Information (NCBI), see Altschul SF et al., "Molecular Biology Journal", 215:403-410 (1990); Stephen F et al., "Nucleic Acids Res.", 25:3389-3402 (1997)), and ClustalW2 (available on the website of the European Bioinformatics Institute, see Higgins DG et al., "Methods in Enzymology", 266:383-402 (1996); Larkin MA et al., "Bioinformatics" (Oxford, England), 23(21):2947-8 This can be achieved using (see 2007) and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can do so by using the default parameters provided by the above tools or by customizing the parameters suitable for matching, such as by selecting an appropriate algorithm. In some embodiments, non-identical residue positions may differ by conservative amino acid substitutions.A "conservative amino acid substitution" is a 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). Generally speaking, conservative amino acid substitutions do not substantially alter the functional properties of a protein. If two or more amino acid sequences differ from each other due to conservative substitutions, the percentage or degree of similarity may be adjusted upward to compensate for the conservative nature of the substitution. Methods for making such adjustments are well known to those skilled in the art. See, for example, Pearson (1994) "Methods Mol. Biol." 24: 307-331, which is incorporated herein by reference.

[0029] "Operatively linked" refers to a functional relationship between two or more polynucleotide sequences. In the context of polynucleotides encoding fusion proteins (e.g., the polypeptide chain of a CAR in this disclosure), the term refers to the joining of these two or more polynucleotide sequences so that the two polynucleotides can be held within the same expression frame and translated into a single amino acid sequence. In the context of transcription or translation regulation, the term refers to a functional relationship between a regulatory sequence and a coating sequence. For example, a promoter is positioned in the correct location and orientation of a coating sequence to regulate transcription. "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. Such means include, but are not limited to, recombinant and synthetic means. The terms "polypeptide" and "protein" can be used interchangeably and refer to a chain of amino acid residues covalently linked by peptide bonds. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0030] "T cell receptors" or "TCRs" refer to protein complexes on the surface of T cells that play a role in recognizing antigen fragments, which are peptides that bind to MHC molecules. The term "chimeric natural killer receptor" (CNK receptor) refers to a composition containing a chimeric NK-activated receptor module and a chimeric NK signaling module. The term "CNKT cell" refers to a T cell that contains the CNK construct. The term "chimeric natural killer receptor-universal T cell" (CNK-UT) refers to T cells that express the NK-activating receptor module construct, the CNK signaling module construct, and the UT module construct. The term "CAR / CNK-UT" refers to chimeric antigen receptor and chimeric natural killer receptor-universal T cells (CNK-UT), which express CAR structures, NK-activated receptor module constructs, CNK signaling module constructs, and UT module constructs that target specific tumor antigens.

[0031] As used herein, the term "costimulatory ligand" includes molecules on antigen-presenting cells (e.g., APCs, dendritic cells, B cells, and other immune cells) that specifically bind to homologous costimulatory molecules on T cells to provide a signal. This signal mediates T cell responses such as proliferation, activation, and differentiation, in addition to primary signals provided, for example, by the binding of the TCR / CD3 complex to MHC molecules containing peptides. Co-stimulatory ligands 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), cell adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin β 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 further include antibodies that specifically bind to costimulatory molecules, particularly those present on T cells, and include, but are not limited to, ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7LIGHT, NKG2C, B7-H3, and CD83. A "costimulatory molecule" or "costimulatory receptor" refers to a congenital binding partner on a T cell that specifically binds to a costimulatory ligand and mediates T cell costimulatory responses, such as proliferation, but is not limited to these. Examples of costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Costimulatory molecules also include non-naturally occurring engineered proteins.

[0032] As used herein, "co-stimulatory signal" refers to a signal that leads to primary signaling, such as TCR / CD3, and results in T cell proliferation and / or upregulation or downregulation of important molecules. The term "stimulus" refers to a primary response induced by the binding of a stimulating molecule (e.g., the TCR / CD3 complex) to its homologous ligand. This mediates a signaling event, for example, through the TCR / CD3 complex. A stimulus can mediate the altered expression of a molecule, such as the downregulation of TGF-B and / or the rearrangement of the cytoskeleton. As used herein, the term "irritant molecule" refers to a molecule that specifically binds to a homologous irritant ligand present on antigen-presenting cells in T cells. As used herein, "irritant ligand" refers to a ligand that, when present on antigen-presenting cells (e.g., APCs, dendritic cells, B cells, etc.), specifically binds to a congenital binding partner on T cells (referred to herein as "irritant molecule") and can mediate primary T cell responses such as activation, initiation of immune response, and proliferation. Irritant ligands are well known in this field and include, in particular, MHC class I molecules containing peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0033] As used herein, "activation" refers to the state of T cells that are sufficiently stimulated to induce detectable cell proliferation. Activation may also relate to the production of induced cytokines and the function of detectable effectors. The term "activated T cell" specifically refers to T cells that are undergoing cell division. "Effective dose" or "therapeutic effective dose" means the amount of cells, compositions, formulations or any material described herein that is effective in achieving the desired biological outcome. Such outcomes include, but are not limited to, the removal of B cells expressing a specific B cell receptor (BCR) and the antibodies produced therefrom. As used herein, “subject” refers to a mammal, such as a human or other animal, and is usually a human. In some embodiments, the subject administered cells, cell populations, or compositions, such as a patient, is a mammal, and is usually a primate, such as a human. In some embodiments, the primate is a monkey or ape. The subject may be male or female and may be of any appropriate age, including infants, adolescents, young adults, adults, and elderly. In some embodiments, the subject may be a non-primate mammal, such as a rodent. As used herein, “treatment” (and its grammatical variations, such as “to treat” or “treating”) means to completely or partially improve or reduce a disease, condition, or disorder, or any symptoms, side effects, consequences, or phenotype associated therewith. Desired therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of disease progression, remission or reduction of the disease state, and remission or improvement of the prognosis. These terms do not imply a complete cure of the disease or the complete elimination of any symptoms or effects of any symptoms or consequences.

[0034] As used herein, “prevention” includes preventing the onset or recurrence of the disease in subjects who are susceptible to the disease but have not been diagnosed with the disease. In some embodiments, the cells and compositions provided are used to slow or delay the progression of the disease. As used herein, "inhibits" a function or activity, meaning that the function or activity is reduced compared to the same conditions (excluding the conditions or parameters of interest) or to other conditions. For example, cells that inhibit tumor growth will reduce the rate of tumor growth compared to when those cells are not present. In the context of administration, the “effective amount” of a reagent (e.g., chimeric antigen receptor, polynucleotide, vector, pharmaceutical preparation, cell, or composition) refers to the amount that effectively achieves the desired outcome, such as therapeutic or preventive results, at the required dosage and time frame. The “therapeutic effective dose” of a reagent (e.g., a pharmaceutical formulation or cells) refers to the amount that effectively achieves the desired therapeutic outcome (e.g., treatment of a disease, condition, or disorder, and / or the pharmacokinetic or pharmacodynamic effect of the treatment) at the required dosage and time. The therapeutic effective dose may vary depending on factors such as the disease state, the subject's age, sex, and weight, and the cell population being administered. In some embodiments, the provided method involves administering cells and / or compositions in an effective dose, e.g., a therapeutic effective dose. The "prophylactic effective dose" refers to the amount of medication that effectively achieves the desired preventive outcome at the required dosage and time. While not always the case, the prophylactic effective dose is usually less than the therapeutic effective dose because prophylactic doses are typically administered to subjects before or in the early stages of the disease. In cases of relatively low tumor burden, the prophylactic effective dose may, in some respects, be higher than the therapeutic effective dose.

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

[0036] The ER-TPD technology platform provided in this application has the following advantages compared to conventional protein degradation / removal technologies (e.g., PROTAC, CRISPR, etc.): 1. ER-TPD chimeric protein constructs are delivered to cells via vectors (e.g., liposomes, nanoparticles, lentiviruses, adenoviruses, oncolytic viruses, etc.), are stably expressed, and exert their function, without the problems of reduced degradation efficiency due to lower cell permeability and shorter half-life compared to PROTAC; 2. ER-TPD technology, based on the ERAD mechanism of the endoplasmic reticulum, directly intervenes in protein synthesis and effectively degrades proteins in the process of protein synthesis and assembly in the endoplasmic reticulum, thus having more effective degradation efficiency compared to exogenous PROTAC technology; 3. While exogenous PROTAC cannot effectively degrade membrane proteins and secreted proteins, ER-TPD directly intervenes in the process of protein synthesis and assembly in the endoplasmic reticulum. ER-TPD technology can directly target, retain, and degrade membrane proteins and secreted proteins, enabling the targeting of many important disease-related target proteins that could not be targeted with conventional TPD technology, thus providing novel drug development rules for disease treatment;4. In the design method of ER-TPD, various single-chain antibody structures or artificial affinity ligand structures can be used as the targeting domain, significantly enhancing the specific and accurate recognition and binding ability to target proteins;On the other hand, conventional PROTAC technology uses the chemical structure of small molecules, making it difficult to ensure the specificity of the targeting structure, so ER-TPD technology has the great advantage of accuracy and specificity of targeting;5. ER-TPD technology can achieve a multi-pathway mechanism of target protein degradation through downstream degradation guide ligand structures. Multi-pathway mechanisms include the UPS mechanism by ubiquitous proteasomes and the ALP mechanism by autophagy lysosomes. By designing downstream degradation guide ligand structures using genetic engineering techniques to flexibly link them to intracellular degradation systems, effective targeted degradation can be achieved.On the other hand, conventional PROTAC technology can only achieve degradation by targeting E3 ligase; 6. ER-TPD technology degrades target proteins at the protein level, and therefore, compared to technologies that block the expression of target proteins at the gene level (e.g., CRISPR technology), it does not have off-target effects or toxicity / side effects such as chromosomal instability due to the gene editing process; 7. ER-TPD technology can be perfectly combined with gene and cell therapy technologies, greatly expanding the clinical indications of current gene and cell therapies and improving clinical efficacy; 8. The development of targeted degradation drugs based on ER-TPD technology is highly feasible and has a short cycle. Nucleic acid and cell therapies based on ER-TPD technology can more easily achieve scale-up and industrial production, and will become an important foundational technology for the development of new drugs.

[0037] In some respects, this disclosure provides a chimeric protein construct using endoplasmic reticulum-based targeted protein degradation (ER-TPD), which comprises an ER-associated degradation (ERAD) mechanism protein-binding domain and a targeting domain. In some embodiments, the chimeric protein construct further includes a proteolytic pathway member (e.g., a ubiquitous-proteasome system pathway member, an endosome-lysosome pathway member, or an autophagy pathway member) binding domain. The proteolytic pathway member binding domain may be linked to a component of the chimeric protein construct by any suitable means, for example, to the ERAD mechanism protein binding domain. In another aspect, the disclosure further provides a library of compositions, each comprising a viral protein or nucleic acid molecule encoding it, comprising a functional motif capable of hijacking the ERAD mechanism to retain it within the ER and accelerating proteasome-mediated degradation, and an affinity moiety that targets and degrades specific proteins, and is used for therapeutic purposes to modify cellular phenotypes, improve cellular function, induce apoptosis, or inhibit viral replication.

[0038] 1. ERAD mechanism protein-binding domain As used herein, the term “ERAD mechanism protein” refers to a protein involved in the ERAD mechanism or pathway. The term “ERAD mechanism protein binding domain” is also called the “ERAD degradation domain” and refers to a portion or domain that binds to and / or is available to an ERAD mechanism protein, such as the transmembrane domain (or its functional variant) and cytoplasmic domain (or its functional variant) of a viral endoplasmic reticulum (ER) commensal glycoprotein, the cytoplasmic domain also called the “endoplasmic reticulum commensal domain” or “ER commensal domain.” In some embodiments, the ERAD mechanism protein-binding domain comprises a transmembrane domain or a functional variant of a viral endoplasmic reticulum (ER) commensal protein, and an ER commensal domain or a functional variant thereof. In some embodiments, the viral ER commensal glycoprotein can be any viral ER commensal protein that can hijack the ERAD mechanism and promote ubiquitination and proteasome-mediated degradation of target proteins.

[0039] i. ERAD mechanism protein-binding domain derived from adenovirus E3-19K In some embodiments, the viral endoplasmic reticulum (ER) commensal protein is adenovirus E3-19K. Adenovirus E3-19K (also known as "E19") comprises three functional modules: a luminal domain that interacts with MHC-I and MICA / B molecules, a transmembrane domain, and an ER commensal domain. Its ER commensal domain contains a dyridine motif in the cytoplasmic tail, which can return the Golgi apparatus to the ER and is therefore also called the "ER return signal motif." Studies have revealed that the transmembrane domain and ER return signal motif are necessary to ensure efficient ER localization, inhibition of transport of histocompatibility complex class I (MHC-I) and MHC-I-associated chains A and B (MICA / B molecules), and degradation of the proteasome. Thus, adenovirus E3-19K can commensalize MHC class I molecules (e.g., MHC-I and MICA / B molecules) in the secretory pathway and interfere with antigen presentation. The chimeric protein constructs provided in this disclosure achieve the objective of degrading target proteins by fusing the transmembrane domain and endoplasmic reticulum (ERAD) domain (i.e., ERAD degradation domain) of the previral E3-19K with the target protein-binding domain. In some preferred embodiments, the ERAD degradation domain of adenovirus E3-19K includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 60, 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. The ERAD degradation domain of adenovirus E3-19K has a dyridine motif in the cytoplasmic tail and can return the Golgi apparatus to the endoplasmic reticulum. In some preferred embodiments, the amino acid sequence of the ERAD degradation domain of adenovirus E3-19K is shown in SEQ ID NO: 60.

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

[0041] a) HCMV US2 and US11 The two proteins HCMV US2 and US11 are both ER-resident type I endogenous membrane glycoproteins that inhibit MHC class I antigen presentation by selecting the ERAD pathway and promoting the degradation of MHC class I heavy chains. Expression of either of these proteins leads to the rapid degradation of newly synthesized MHC class I heavy chains. US2 and US11 bind to MHC class I heavy chains via their luminal domains and recruit host cell proteins, which extract polypeptides from the endoplasmic reticulum membrane by "pulling" the cytoplasmic tail of the heavy chain. After translocation to the cytoplasm, the MHC class I molecules are ubiquitinated and degraded by the proteasome. In addition to class I molecules, US2 also causes degradation of two proteins in the class II pathway, DR-α and DM-α, as well as HFE (a non-standard major histocompatibility complex (MHC) class I protein involved in iron regulation). The luminal domain of US2 is responsible for binding to MHC class I and class II molecules, while the transmembrane domain (TM) and cytoplasmic domain (CT) interact with cellular components of the ERAD mechanism or pathway, contributing to translocation and the promotion of enzymatic degradation of two 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), and is required for US2-dependent MHC heavy chain translocation (see Joana et al., Signal peptide peptidase is required for dislocation from the endoplasmic reticulum. Nature volume 441, pages 894-897 (2006)). Furthermore, US2 interacts with the endoplasmic reticulum-resident RING-type E3 ligase TRC8 through its TM domain, thereby contributing to the ubiquitination and proteasome degradation of MHC I and II molecules anchored by the US2 tail (Stagg HR et al., 2009).

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

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

[0044] b) HCMV US3 The HCMV US3 glycoprotein does not promote the degradation of MHC proteins, but rather physically binds to MHC class I heterodimers containing peptides, retaining the class I complex in the ER and inhibiting the binding of the immutable chain to the class IIDR-αβ dimer in the ER, leading to mislocalization of the class II complex and reduced peptide loading. Therefore, US3 can interfere with the intracellular transport and maturation of MHC class I molecules in the early stages of HCMV infection. US3 is a commensal membrane protein of the endoplasmic reticulum. Domain exchange experiments revealed that the luminal domain of US3 allows US3 itself to retain the endoplasmic reticulum, and that both the luminal and transmembrane domains are essential for the retention of class I MHC molecules in the endoplasmic reticulum. In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US3 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 51, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, and possesses endoplasmic reticulum commensal function. In some embodiments, the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US3 is shown in SEQ ID NO: 51. In some preferred embodiments, the ERAD degradation domain of US3 comprises an amino acid sequence having 80% or more identity with 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 more identity, and more preferably an amino acid sequence having 98% or 99% or more identity, and has endoplasmic reticulum commensal function. In some embodiments, the amino acid sequence of the ERAD degradation domain of US3 is shown in SEQ ID NO: 79.

[0045] c) HCMV US10 The HCMV US10 glycoprotein also interacts with components of MHC class I antigen presentation. US10 binds to free class I heavy chains and slows their transport from the ER. However, US10 does not affect US2 or US11. In some preferred embodiments, the ERAD degradation domain 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: 57, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, and possesses endoplasmic reticulum commensal function. In some embodiments, the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US10 is shown in SEQ ID NO: 57.

[0046] d) HCMV US6, HSV ICP47, CPXV012, EBV BNFL2a, BHV UL49.5 Unlike US2, US3, US10, and US11, the HCMV L protein US6 influences antigen presentation through a completely different strategy. Instead of interacting with free class I heavy chains or fully assembled class I complexes, US6 inhibits cytoplasmic peptide transposition via the TAP complex (TAP1 / 2). US6 prevents ATP binding by binding to the ER luminal side of TAP1 and inducing a structural change. Residues 89-108 in the ER-luminal domain of US6 contribute to the binding of US6 to TAP and are necessary conditions for this inhibition. Such inhibition of TAP activity affects not only the expression of standard MHC class I alleles but also the expression of non-standard alleles HLA-C and HLA-G in embryonic trophoblast cells. HSV ICP47, expressed as the HCMV US6 protein early in the infection cycle, is optional for in vitro replication, and a similar strategy can be applied to block the assembly of class I molecules. ICP47 inhibits TAP-mediated peptide transport and binds tightly to the TAP1-TAP2 complex. One clue to the mechanism by which ICP47 inhibits TAP is its high species selectivity. Both HSV1 and HSV2I CP47 inhibit TAP in gorillas, monkeys, pigs, dogs, and cattle, while having little effect on TAP in mice, rats, guinea pigs, or rabbits. ICP47 has approximately 100 times higher affinity for human TAP than for mouse TAP. ICP47 inhibits the binding of peptides to TAP but does not affect ATP binding. ICP47 has an affinity for TAP that is 10 to 1000 times higher than most peptides, and is thought to be used as a competitive inhibitor of peptide-TAP binding, and to bind directly to the peptide binding site.

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

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

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

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

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

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

[0053] 2. Targeted Domains The targeting domain (target protein binding domain) of the chimeric protein construct provided in this disclosure may be any structure that recognizes and binds to the target protein, such as an antibody, antibody fragment, a functional motif derived from a natural protein (e.g., a natural ligand), a synthetic polypeptide or protein having affinity for the target protein, or various variants of the above molecules, such as variants, fusions, or cleaved products. For example, the targeting domain may be a natural ligand of the target protein, an antibody that specifically recognizes the target protein or an antigen-binding fragment thereof, or an antigen that is specifically recognized by the antibody. In some embodiments, the targeting domain includes antibodies or functional fragments thereof that specifically target a target protein (e.g., diabodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide bond-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide bond-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), Fv(scFv), multispecific antibodies (e.g., bispecific antibodies), camelized single-domain antibodies (e.g., VHH), nanobodies, domain antibodies, bivalent domain antibodies, double-chain antibodies, triple-chain antibodies, and quadruple-chain antibodies). The target protein domain may include, but is not limited to, any binding ligand capable of binding to the target protein, such as a ligand, a ligand-binding portion in a receptor, or a host protein-binding portion in a viral protein.

[0054] The target protein may be any protein whose level or activity regulation is desired. For example, by altering its activity via the ERAD mechanism, it blocks proteins within the ER and accelerates proteasome-mediated degradation. Such degradation can achieve therapeutic objectives (e.g., improved therapeutic effect, reduced side effects) by altering the cellular phenotype, enhancing cellular function, inducing apoptosis, and / or suppressing viral replication. In some embodiments, the target protein may be a protein involved in the cell cycle, apoptosis, signaling, cell differentiation, cell dedifferentiation, cell growth, production of cytokines or their biomodulators, production of regulatory or functional proteins, pro-inflammatory signaling, or glucose regulatory pathways. In another embodiment, the target protein is a disease-related protein (also called a “pathogenic protein”), and as used herein, “disease-related protein” or “pathogenic protein” refers to a protein whose functional or activity changes lead to the onset of a disease, or whose function is relatively important for the progression of a disease. Exemplary pathogenic proteins include, but are not limited to, oncogenic proteins, viral proteins, and proteins that cause autoimmune diseases (e.g., antibodies produced by plasma cells). In some embodiments, the target protein is selected from immune function-related target proteins, neurological disease-related target proteins, infectious disease-related target proteins (e.g., viral infection-related target proteins), autoantigen-related target proteins or tumor-related target proteins (or oncogenic proteins), and metabolic disease-related target proteins.

[0055] In some embodiments, the pathogenic protein is an oncogenic protein. The oncogenic protein may be encoded by an oncogene. Examples of such oncogenes include 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, and BRCA2. , CBL, CCND1, CDKN1A, CDKN1C, CDKN2A, CDKN2B, CRK, CRK-II, CSF1R / FMS, DBL, DDOST, PMS-2, PRAD-1, RAF, RHOM-1, RHOM-2, SIS, TAL2, TANI, TI AM1, TSC2, TRK, TSC1, STK11, PTCH, MEN1, MEN2, P57 / KIP2, PTEN, HPC1, ATM, XPA / XPG, BCL6, DEK, AKAP13, CDH1, BLM, EWSR1 / FLI1, FES, FGF3, FE R, FGR, FLI1 / ERGB2, FOS, FPS / FES, FRA1, FRA2, FYN, HCK, HEK, HER3 / ERBB-2, ERBB-3, HER4 / ERBB-4, HST2, INK4A, INK4B, JUN, JUNB, JUND, KIP2 , KIT, KRAS2A, KRAS2B, LCK, LYN, MAS, MAX, MCC, MLH1, MOS, MSH2, MYBA, MYBB, NF1, NF2, P53, PDGFB, PIM1, PTC, RBI, RET, ROS1, SKI, SRC1, TALI, TGFBR2, THRA1, THRB, TIAM1, TRK, VAV, VHL, WAF1, WNT2, WT1, YES1, ALK / NPM1, AMI1, AXL, FMS, GIP, GLI, GSP, HOX11, HST, IL3, INT2, KS3, K-SAM , LBC, DCC, DPC4 / SMAD4, E-CAD, E2F1 / RBAP, ELK1, ELK3, EPH, EPHA1, E2F1, EPHA3, ERG, ETS1, ETS2, LMO-1, LMO-2, L-MYC, LYL1, LYT-10, MDM-2,MLH1, MLL, MLM, N-MYC, OST, PAX-5, PMS-1, FGF4, FGF6, FANCA, FLI1 / ERGB2, FOSL1, FOSL2, GLI, HRAS1 , HRX / MLLT1, HRX / MLLT2, KRAS2, MADH4, MASI, MCF2, MLLT1 / MLL, MLLT2 / HRX, MTG8 / RUNX1, MYCLK1, MY H11 / CBFB, NFKB2, NOTCH1, NPM1 / ALK, NRG / REL, NTRK1, PBX1 / TCF3, PML / RARA, PRCA1, RUNX1, RUNX1 / C BFA2T1, SET, SHP2, TCF3 / PBX1, TNFa, Clusterin, Survivin, TOEβ, c-fos, c-SRC and INT-1 is one example, but it is not limited to these. In some embodiments, the oncogenic proteins include Bcl-2 family members (e.g., Bcl-2, Bcl-xL, and Bcl-w), VEGF / VEGFR, PDGFRβ, EGFR, EGFR variants, IGF-1R, HDACs, HER2, MYC, KRAS, AFP, CEA, CA199, estrogen receptor (ER-α), androgen receptor (AR), tyrosine kinases (c-ABL, BCR-ABL, BTK, FAK, PTK6, Wee1, TRK transmembrane receptor), and 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), and DPP3 may be present.

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

[0057] In some embodiments, the target protein includes a viral infection-associated target protein. Examples of the aforementioned virus infection-related target proteins include HBV code 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 novel coronavirus RNA-dependent RNA polymerase, herpesviruses DNA, RNA polymerase, herpesvirus capsid glycoprotein, CMV DNA polymerase, CMV capsid glycoprotein, RSV envelope protein, RSV capsid protein, RSV RNA polymerase, influenza virus RNA polymerase, influenza virus envelope protein, and HPV. Examples include DNA polymerase and 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, or HIV Env protein, whose function is considered important for viral replication, proliferation, function, and the progression of viral disease.

[0058] In some embodiments, the target protein includes an autoantigen-related target protein. In this field, many autoantigens associated with autoimmune diseases are known, including, for example, autoantigens associated with type 1 diabetes such as pancreatic islet cell antigen (ICA), insulin (IAA), glutamate 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 shock protein (HSP60), BiP, PGK1, stress-induced phosphoprotein 1, and FUSE-BP1 / 2; and autoantigens associated with rheumatoid arthritis (RA) such as deoxyribonucleoprotein, SmD1, SmD3, Clq, lupus anticoagulant (LA), cardiolipin (CL), and β2 glycoprotein I (β2 GP). I) Systemic lupus erythematosus (SLE)-related autoantigens such as prothrombin (PT) and phosphatidylserine (PS); Systemic sclerosis (SSc) / scleroderma (SD)-related autoantigens such as Scl-70, SSA, Ro52; Mitochondrial antigens, autoimmune liver disease-related antigens such as Spl00, PML, gp210, p62; Myasthenia gravis-related autoantigens such as acetylcholine receptors; 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 Autoantigens associated with central nervous system autoimmune diseases (limbic encephalitis, encephalomyelitis, cerebellar ataxia), such as (GABAB) receptors and 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-induced irritable bowel syndrome, such as endomysial antibodies (EMA) and tissue transglutaminase (tTG);These include anti-NMDAR antibody autoantigens such as N-methyl-D-aspartate receptors (NMDARs), autoantigens associated with neuromyelitis optica (NMO) such as aquaporin 4 (AQP4), and reproductive-related autoantigens such as ovarian antigens and sperm antigens. In some embodiments, the pathogenic protein is a metabolic disease-related target protein. Examples of metabolic disease-related target proteins include, but are not limited to, atherosclerosis (AS)-related target proteins such as CD36, low-density lipoprotein receptor (LDLR), ChemR23 (CMKLR1), and mitochondrial dehydrogenase (ALDH4A1) (and its target proteins); but are not limited to, type II diabetes-related target proteins such as RalGAPα1 and dipeptidyl peptidase IV (DPP4); but are not limited to, non-alcoholic fatty liver-related target proteins such as TMEM16A and VAMP3; but are not limited to, tumor glucose metabolism-related target proteins such as hexokinase (HK), glucose transporter 1 (GLUT1), glucose transporter 4 (GLUT4), phosphoglycerate dehydrogenase, and lactate dehydrogenase; but are not limited to, ATP citrate lyase. Tumor lipid metabolism-related target proteins include lyase (ACLY), fatty acid synthase (FASN), and cholesterol esterase (acetyl-CoA acetyltransferase 1, ACAT1).

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

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

[0061] In some preferred embodiments, the binding protein molecular domain or functional variant thereof that targets MHC I and / or MHC II is derived from a viral glycoprotein that degrades MHC and / or MHC II molecules; preferably, the viral glycoprotein is selected from HCMV glycoproteins US2, US3, US11 or US10, adenovirus E3-19K or HHV-7 US21. In some preferred embodiments, the MHC I and / or MHC II targeting binding protein molecular domain or a functional variant thereof further comprises a viral protein that directly inhibits or degrades the NK target protein MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, or ULBP6; preferably, the viral protein is selected from HCMV UL16, UL141, UL142, or adenovirus E3-19K. In some preferred embodiments, the MHC I and / or MHC II targeting binding protein molecular domain or a functional variant thereof further comprises a viral protein that transports and degrades MHC I molecules from the Golgi apparatus to lysosomes; preferably, the viral protein is selected from HIV Nef, HIV Vpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3, and HTLV-1 p12. In some preferred embodiments, the MHC I and / or MHC II targeting binding protein molecular domain or a functional variant thereof 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. In some preferred embodiments, the MHC-binding domain of HHV-7 US21 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 62, 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; the amino acid sequence of the MHC-binding domain of HHV-7 US21 is shown in SEQ ID NO: 62.

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

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

[0064] Members of the endosomal-lysosome 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. Examples of autophagy degradation pathway members include chaperone-mediated autophagy (CMA), USP10, G3BP1, ULK1, ATG16L1, TRIM16, FBXO27VDAC, RHOT1, MFN1 / 2, BNIP3L, FUNDC1, BNIP3, AMBRA1, BCL2LI3, FKBP8, CHDH, DISC1, PHB2, cardiolipin, SEC62, RTN3, PEX5, PEX14, ABCD3, and NUFIP1. In some embodiments, the proteolytic pathway member binding domain is a VHL binding domain. In some embodiments, the VHL binding domain comprises the amino acid sequence of SEQ ID NO: 119 (DRHDSGLDSM) or SEQ ID NO: 120 (ALAPYIP). In some embodiments, the proteolytic pathway member binding domain is a Keap1 binding domain. In some embodiments, the Keap1 binding domain includes the amino acid sequence of SEQ ID NO: 121 (LDPETGEYL). In some embodiments, the proteolytic pathway member binding domain is an E3 ubiquitin ligase binding domain. The E3 ubiquitin ligase binding domain contains the amino acid sequence of Sequence ID No. 119 (DRHDSGLDSM). In some embodiments, the proteolytic pathway member binding domain is a CMA binding domain. In some embodiments, the CMA binding domain includes the amino acid sequence of SEQ ID NO: 122 (KFERQ). In some embodiments, the CMA binding domain includes the amino acid sequence of SEQ ID NO: 123 (KFERQKILDQRFFE).

[0065] In some embodiments, the proteolytic pathway member binding domain is a proteasome binding domain. In some embodiments, the proteasome binding domain is selected from the ubiquitin-like (UbL) domain of yeast Rad23 (e.g., S. cerevisiae Rad23), 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. The inventors of this application have found that chimeric protein constructs containing a protein degradation pathway member binding domain (e.g., E3L, E2L, or lysosome ligand) (LL) (for example, those described above) significantly improve the degradation effect on target proteins compared to the basic TPD design (Targeted protein binding domain-Transmembrane domain-ER retention domain, TBD-TMD-ERD). Functional structures derived from viral ER retention proteins differ in their binding ability to the ERAD reverse transporter complex, and the native structures also differ in their interaction with ER retention E3 ligases and compete with the normal degradation pathway of misfolded proteins in the endoplasmic reticulum's ERAD mechanism. Therefore, the domains of natural TMD-ERDs exhibit differences and limitations in ubiquitination and degradation of target proteins. To further improve the efficiency of target protein degradation, introducing ubiquitosis-proteasome system (UPS) ligand domains and / or autophagy-lysosome pathway (ALP) ligand domains can further promote ubiquitination and proteasomal degradation of target proteins transported into the cytoplasm, or transport them to lysosomes to achieve effective hydrolysis of target proteins. In some embodiments, the targeting domain or ERAD mechanism protein-binding domain of the chimeric protein construct is linked to a proteolytic pathway member-binding domain by a hinge or linker. The hinge or linker may be one or more molecular sequence structures (Glyx ​​Sery)n, where n may be a number from 1 to 10, and x and y may each be a number from 0 to 10, provided that x and y are not simultaneously 0. In some embodiments, the hinge may include the amino acid sequence shown in SEQ ID NO: 172 (IgG4 hinge) or SEQ ID NO: 173 ((Gly4Ser)2).

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

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

[0068] 5. Co-expression portion In some embodiments, the chimeric protein construct provided in this disclosure and at least one co-expression moiety are expressed together. Any means suitable for co-expression can be used. For example, the chimeric protein construct (or a portion thereof) and the at least one co-expression moiety may be linked by a cleavable linker, and when the linker is cleaved, the chimeric protein construct and the co-expression moiety are expressed together. In some embodiments, the chimeric protein construct provided in this disclosure and at least one co-expression moiety are linked, for example, by a self-cleavable linkage. In some embodiments, the self-cleavable linkage is a cleavable peptide such as a T2A peptide, GSG-T2A peptide, E2A peptide, GSG-E2A peptide, F2A peptide, GSG-F2A peptide, P2A peptide, or GSG-P2A peptide. In some preferred embodiments, the T2A comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 108, 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; the amino acid sequence of the T2A is shown in SEQ ID NO: 108. In some preferred embodiments, the amino acid sequence of the GSG-T2A peptide is shown in SEQ ID NO: 109. In some preferred embodiments, P2A comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 110, 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; the amino acid sequence of P2A is shown in SEQ ID NO: 110. In some preferred embodiments, the amino acid sequence of the GSG-P2A peptide is shown in SEQ ID NO: 111. In some preferred embodiments, E2A comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 112, 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; the amino acid sequence of E2A is shown in SEQ ID NO: 112.

[0069] In some preferred embodiments, the amino acid sequence of the GSG-E2A peptide is shown in SEQ ID NO: 113. In some preferred embodiments, 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%, or 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. In some preferred embodiments, the amino acid sequence of the GSG-F2A peptide is shown in SEQ ID NO: 115. The co-expression moiety provided in this application may be any protein or polypeptide having a biological function. An appropriate co-expression moiety can be selected depending on the desired biological function. For example, a protein that degrades or reduces MHC class I or class II molecules can be selected as the co-expression moiety to reduce cellular immunogenicity or antigen presentation. Alternatively, a binding domain that recognizes or binds to a target protein (e.g., a chemokine receptor or chimeric antigen receptor CAR) can be selected as the co-expression moiety to enable cells to recognize the target protein. Furthermore, an immunostimulatory molecule can be selected as the co-expression moiety to enhance immunostimulatory activity. In some preferred embodiments, examples of the co-expressed moieties include intact viral ER commensal glycoproteins (e.g., HCMV US2, US3, US11, US10, adenovirus E3-Kl9, 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 activation receptors (e.g., NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, and native cytotoxic receptors). Examples include, but are not limited to, steroids (TRAIL, DNAM-1, CD16a, 2B4, NTB-A, CRACC, and NKp80), CNK signaling modules, cytokines, CD7, and immunostimulatory molecules (e.g., one or more selected from TNF-α, IFN-β, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, and granulocyte-macrophage colony-stimulating factors).

[0070] i) intact viral ER commensal glycoproteins In some embodiments, the co-expressed moiety is an intact viral ER commensal glycoprotein, and includes, but is not limited to, HCMV US2, US3, US11, US10, adenovirus E3-Kl9, HCMV US6, and HSV ICP47. In some preferred embodiments, the full-length sequence of the HCMV glycoprotein US2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 46, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, and preferably has the activity to degrade MHC class I or class II molecules; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US2 is shown in SEQ ID NO: 46. In some preferred embodiments, the full-length sequence of US2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 74, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, and preferably has the activity to degrade MHC class I or class II molecules; the amino acid sequence of the full-length sequence of US2 is shown in SEQ ID NO: 74. In some preferred embodiments, the full-length sequence of the HCMV glycoprotein US3 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 49, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, and preferably has the activity to degrade MHC class I or class II molecules; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US3 is shown in SEQ ID NO: 49. In some preferred embodiments, the full-length sequence of US3 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 77, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, and preferably has the activity to degrade MHC class I or class II molecules; the amino acid sequence of the full-length sequence of US3 is shown in SEQ ID NO: 77.

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

[0072] ii) Chimeric antigen receptor (CAR) In some embodiments, the co-expressed portion is a chimeric antigen receptor (CAR). The CAR comprises a target protein-binding domain (e.g., an extracellular recognition domain that targets a tumor), a transmembrane domain, and an immune receptor-activated signaling domain (ITAM) (also known as the “intracellular signaling domain”). In some embodiments, the CAR may further include a co-stimulatory domain. In some embodiments, a hinge or linker is included between the extracellular recognition domain that targets the tumor, the transmembrane domain, and / or the intracellular signaling domain. In some embodiments, the extracellular recognition domain that targets the tumor is selected from a tumor antigen-specific binding domain, a tumor microenvironment-targeting antigen-binding domain, and / or a chemokine receptor that targets the tumor microenvironment. In some preferred embodiments, the extracellular recognition domain that targets the tumor is selected from an antibody or a functional fragment thereof that targets and recognizes a tumor-associated antigen, a TCR, or a combination thereof; the functional fragment of the antibody is selected from Fd, Fv, Fab, Fab', F(ab')2, Fv(scFv), a single-chain antibody (scFv) or nanobody, a double-chain antibody, a triple-chain antibody, and a quadruple-chain antibody.

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

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

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

[0076] In some embodiments, the CAR hinge and / or transmembrane domain described herein provides cell surface presentation of the extracellular domain of the CAR. The CAR hinge described herein may be derived from any membrane-bound protein or transmembrane protein, such as BAFFR, BLAME (SLAMF8), CD2, CD3ε, CD4, CD5, CD8, CD9, CD11a (CD18, ITGAL, LFA-1), CD11b, CD11c, CD11d, CD16, CD19, CD22, CD27, CD28, CD29, CD33, CD37, CD40, CD45, CD49a, CD49d, CD 49f, CD64, CD80, CD84, CD86, CD96(Tactile), CD100(SEMA4D), CD103, CD134, CD137(4-1BB), CD150(IPO-3, SLAMF1, SLA M), CD154, CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(Ly9), CD244(2B4, SLAMF4), CD278(ICOS), CEACAM1, CRT AM, GITR, HYEM (LIGHTR), IA4, IL2Rβ, IL2Rγ, IL7Ra, ITGA1, ITGA4, ITGA6, ITGAD, ITGAE, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIR, LTBR, ​​OX40, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG / Cbp, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, α, β, or ζ chains of T cell receptors, TNFR2, VLA1, and VLA-6 are among the examples, but are not limited to these. In some embodiments, the CAR hinge described herein includes the hinge region of CD8α, the hinge region of human immunoglobulin (Ig), or a glycine-serine-rich sequence. In some preferred embodiments, the linker is a flexible linker; preferably, the flexible linker comprises the amino acid sequence (Gly(x)Ser(y))n, where n is an integer from 1 to 10, and x and y are independently integers from 0 to 10, provided that both x and y are not 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.

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

[0078] In some preferred embodiments, the T2A comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 108, 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; the amino acid sequence of the T2A is shown in SEQ ID NO: 108. In some preferred embodiments, the amino acid sequence of the GSG-T2A peptide is shown in SEQ ID NO: 109. In some preferred embodiments, P2A comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 110, 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; the amino acid sequence of P2A is shown in SEQ ID NO: 110. In some preferred embodiments, the amino acid sequence of the GSG-P2A peptide is shown in SEQ ID NO: 111. In some preferred embodiments, E2A comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 112, 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; the amino acid sequence of E2A is shown in SEQ ID NO: 112. In some preferred embodiments, the amino acid sequence of the GSG-E2A peptide is shown in SEQ ID NO: 113. In some preferred embodiments, 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%, or 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. In some preferred embodiments, the amino acid sequence of the GSG-F2A peptide is shown in SEQ ID NO: 115.

[0079] iii) CNK receptor In some embodiments, the co-expressed portion is a CNK receptor. The CNK receptor comprises a chimeric NK-activating receptor module and a chimeric NK signaling module (also referred to as the "CNK signaling module"). The chimeric NK-activating receptor module may include at least an NK cell-activating receptor or a functional variant thereof. NK cell activating receptor In some embodiments, the NK cell activating receptor comprises (a) the extracellular domain (ED) or a functional variant thereof of the NK cell activating receptor, (b) the transmembrane domain (TMD) or a functional variant thereof of the NK cell activating receptor, and (c) the intracellular domain (ICD) or a functional variant thereof of the NK cell activating receptor, wherein a hinge or linker may be included between the extracellular domain or a functional variant thereof of the NK cell activating receptor, the transmembrane domain or a functional variant thereof of the NK cell activating receptor, and / or the intracellular domain or a functional variant thereof of the NK cell activating receptor. For further details of the NK cell activating receptor and the CNK signaling module, see, for example, US 2020 / 0308248 Al, the entire contents of which are incorporated herein by reference. The NK cell activation receptors are selected from NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, Natural Cytotoxicity Receptors (NCR), TRAIL, DNAM-1, Signaling Lymphocyte Activation Molecule (SLAM) family molecule 2B4 (also known as CD244), DNAX accessory molecule 1 (also known as DNAM-1, CD226), CD16a, 2B4, NTB-A, CRACC(CS1), and NKp80; where the Natural Cytotoxicity Receptors include 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 innate cytotoxic receptor is selected from NKp46, NKp44, and NKp30.

[0080] In some preferred embodiments, the NK cell activating receptor is a mammalian NK cell activating receptor; preferably, the mammal is selected from humans, primates, mice, horses, cattle, sheep, goats, cats, pigs, dogs, llamas, alpacas, elephants, squirrels, and guinea pigs. In some preferred embodiments, the NK cell activation receptor is a recombinant NK cell activation receptor comprising NK cell activation receptor domains of different origins. In some preferred embodiments, the NK cell activating receptor is a human-derived NK cell activating receptor; preferably, the NK cell activating receptor is a recombinant NK cell activating receptor comprising different human-derived NK cell activating receptor domains. In some preferred embodiments, the NK cell activation receptor is a mouse-derived NK cell activation receptor; preferably, the NK cell activation receptor is a recombinant NK cell activation receptor comprising different mouse-derived NK cell activation receptor domains. In some preferred embodiments, the NK cell activation receptor is a recombinant NK cell activation receptor comprising human and mouse-derived NK cell activation receptor domains. In some preferred embodiments, the extracellular domain of the NK cell activating receptor is the extracellular domain of a human or mouse NK cell activating receptor. In some preferred embodiments, the transmembrane domain of the NK cell activating receptor is the transmembrane domain of a human or mouse NK cell activating receptor. In some preferred embodiments, the intracellular domain of the NK cell activating receptor is the intracellular domain of a human or mouse NK cell activating receptor. In some preferred embodiments, the NK cell activating receptor comprises the extracellular domain of a human NK cell activating receptor, the transmembrane domain of a mouse NK cell activating receptor, and the intracellular domain of a human NK cell activating receptor. In some preferred embodiments, the functional variant of the NK cell activating receptor is selected from a variant of the NK cell activating receptor, a wild-type fusion protein, or a fusion protein of the wild-type and the mutant. In some preferred embodiments, the extracellular domain of human NKG2D comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity; the amino acid sequence of the extracellular domain of human NKG2D is shown in SEQ ID NO: 1.

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

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

[0083] In some preferred embodiments, the full-length sequence of human KIR2DS1 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 11, 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; the amino acid sequence of the full-length sequence of human KIR2DS1 is shown in SEQ ID NO: 11. In some preferred embodiments, the full-length sequence of human KIR2DS2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 12, 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; the amino acid sequence of the full-length sequence of human KIR2DS2 is shown in SEQ ID NO: 12. In some preferred embodiments, the full-length sequence of human KIR2DS4 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 13, 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; the amino acid sequence of the full-length sequence of human KIR2DS4 is shown in SEQ ID NO: 13. In some preferred embodiments, the full-length sequence of human KIR3DS1 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 14, 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; the amino acid sequence of the full-length sequence of human KIR3DS1 is shown in SEQ ID NO: 14.

[0084] In some preferred embodiments, the full-length sequence of human NKp46 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 15, 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; the amino acid sequence of the full-length sequence of human NKp46 is shown in SEQ ID NO: 15. In some preferred embodiments, the full-length sequence of human NKp44 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 16, 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; the amino acid sequence of the full-length sequence of human NKp44 is shown in SEQ ID NO: 16. In some preferred embodiments, the full-length sequence of human NKp30 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 17, 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; the amino acid sequence of the full-length sequence of human NKp30 is shown in SEQ ID NO: 17. In some preferred embodiments, the full-length sequence of human DNAM1 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 18, 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; the amino acid sequence of the full-length sequence of human DNAM1 is shown in SEQ ID NO: 18. In some preferred embodiments, the full-length sequence of human TRAIL comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 19, 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; the amino acid sequence of the full-length sequence of human TRAIL is shown in SEQ ID NO: 19.

[0085] In some preferred embodiments, the full-length sequence of human CD16a comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 20, 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; the amino acid sequence of the full-length sequence of human CD16a is shown in SEQ ID NO: 20. In some preferred embodiments, the full-length sequence of human 2B4 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 21, 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; the amino acid sequence of the full-length sequence of human 2B4 is shown in SEQ ID NO: 21. In some preferred embodiments, the full-length sequence of human NTB-A comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 22, 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; the amino acid sequence of the full-length sequence of human NTB-A is shown in SEQ ID NO: 22. In some preferred embodiments, the full-length sequence of human CRACC comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 23, 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; the amino acid sequence of the full-length sequence of human CRACC is shown in SEQ ID NO: 23. In some preferred embodiments, the full-length sequence of human NKp80 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 24, 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; the amino acid sequence of the full-length sequence of human NKp80 is shown in SEQ ID NO: 24.

[0086] CNK signaling module In some embodiments, the CNK signaling module includes at least (i) an NK cell signaling adapter (e.g., DAP10 or DAP12) or a functional variant thereof. In some embodiments, the NK cell signaling adapter includes (a) an extracellular domain (ED) or a functional variant thereof of the NK cell signaling adapter, (b) a transmembrane domain (TMD) or a functional variant thereof of the NK cell signaling adapter, and (c) an intracellular domain (ICD) or a functional variant thereof of the NK cell signaling adapter; a hinge or linker may be included between the extracellular domain or a functional variant thereof of the NK cell signaling adapter, the transmembrane domain or a functional variant thereof of the NK cell signaling adapter and / or the intracellular domain or a functional variant thereof of the NK cell signaling adapter. In some preferred embodiments, the NK cell signaling adapter is a mammalian NK cell signaling adapter; preferably, the mammal is selected from humans, primates, mice, horses, cattle, sheep, goats, cats, pigs, dogs, llamas, alpacas, elephants, squirrels, and guinea pigs. In some preferred embodiments, the NK cell signal adapter is a recombinant NK cell signal adapter comprising NK cell signal adapter domains of different origins. In some preferred embodiments, the NK cell signal adapter is a human-derived NK cell signal adapter; preferably, the NK cell signal adapter is a recombinant NK cell signal adapter comprising different human-derived NK cell signal adapter domains. In some preferred embodiments, the NK cell signal adapter is a mouse-derived NK cell signal adapter; preferably, the NK cell signal adapter is a recombinant NK cell signal adapter comprising different mouse-derived NK cell signal adapter domains.

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

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

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

[0090] In some preferred embodiments, the human CD3zeta intracellular signaling domain sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 32, 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; the amino acid sequence of the human CD3zeta intracellular signaling domain sequence is shown in SEQ ID NO: 32. In some preferred embodiments, the human DAP10-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 33, 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; the amino acid sequence of the human DAP10-CD3zeta sequence is shown in SEQ ID NO: 33. In some preferred embodiments, the human DAP12-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 34, 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; the amino acid sequence of the human DAP12-CD3zeta sequence is shown in SEQ ID NO: 34. In some preferred embodiments, the human DAP10-DAP12-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 35, 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; the amino acid sequence of the human DAP10-DAP12-CD3zeta sequence is shown in SEQ ID NO: 35. In some preferred embodiments, the human 41BB intracellular signaling domain sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 36, 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; the amino acid sequence of the human 41BB intracellular signaling domain sequence is shown in SEQ ID NO: 36.

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

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

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

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

[0095] i) Modification of nucleic acid molecules (e.g., mRNA) Naturally occurring eukaryotic mRNA molecules may contain stabilizing elements. These stabilizing elements include, but are not limited to, the 5'-terminal untranslated region (UTR) (5' UTR) and / or the 3'-terminal UTR (3' UTR), as well as other structural features such as the 5'-cap structure and the 3'-poly-A tail. Both the 5' UTR and the 3' UTR are normally transcribed from genomic DNA and are part of premature mRNA. Characteristic structures of mature mRNA (e.g., the 5'-cap structure and the 3'-poly-A tail) are usually added to the transcribed (premature) mRNA during the mRNA processing process. The "5' UTR" is a region located immediately upstream (i.e., 5') of the mRNA start codon (i.e., the first codon of the mRNA transcript translated into a ribosome) and does not encode a polypeptide. During RNA transcript generation, the 5' UTR may contain a single promoter sequence. This promoter sequence is well known in the art. It should be understood that such a promoter sequence is not present in the nucleic acid molecules of this disclosure. The "3' UTR" is a region located immediately downstream (i.e., at 3') of the mRNA stop codon (i.e., the codon in the mRNA transcript that signals the termination of translation), and it does not encode a polypeptide.

[0096] The "poly-A tail" is the downstream region of mRNA, for example, immediately downstream of the 3' UTR (i.e., 3'), and contains multiple consecutive adenosine monophosphates. A single 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 the relevant biological environments (e.g., intracellular or in vivo), the function of the poly(A) tail is to protect mRNA from enzymatic degradation (e.g., enzymatic degradation in the cytoplasm) and to contribute to transcription termination and / or the transport and translation of mRNA from the cell nucleus. The 3'-poly(A) tail is an adenine nucleotide typically attached to the 3' end of transcribed mRNA and may contain approximately 400 adenine nucleotides. The length of the 3'-poly(A) tail can affect the stability of the mRNA molecule itself. 5'-capping of polynucleotides can be completed simultaneously with the transcription reaction in vitro, and 5'-guanosine cap structures can be generated using the following chemical RNA cap analogues: 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 Bio Laboratory, Ipswich, Massachusetts) according to the manufacturer's protocol. 5'-capping of modified RNA can be completed after transcription using cowpox virus capping enzyme, generating the "cap 0" structure: m7G(5')ppp(5')G (New England Bio Laboratory, Ipswich, Massachusetts). Cap 1 structure: m7G(5')ppp(5')G-2'-O-methyl can be generated using cowpox virus capping enzyme and 2'-O methyltransferase. Cap 2 structure can be generated from Cap 1 structure, and then the 5'-third-to-last (5'-antepenultimate) nucleotide is 2'-O-methylated using 2'-O methyltransferase. Cap 3 structure can be generated from Cap 2 structure, and then the 5'-fourth-to-last (5'-preantepenultimate) nucleotide is 2'-O-methylated using 2'-O methyltransferase. Recombinant enzymes may also be used. mRNA molecules containing at least one of the above-mentioned stabilizing elements can significantly increase the expression level of the protein they encode. Furthermore, when two or more of the above-mentioned stabilizing elements are used, the expression level of the protein shows a synergistic effect compared to mRNA using a single stabilizing element.

[0097] In some embodiments, the mRNA described herein has one or more AU-rich sequences removed. AU-rich sequences, also known as AURES, are destabilizing sequences found in the 3' UTR. In some embodiments, the mRNA described herein further comprises an open reading frame (ORF) encoding a signal peptide. The signal peptide may comprise the 15th to 60th amino acids at the N-terminus of the protein and, as it is typically required for membrane transport across secretory pathways, generally controls the secretory pathways of many proteins to eukaryotes and prokaryotes. The length of the signal peptide may consist of 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, or 59 amino acids. An "open reading frame" is a continuous sequence of DNA that begins with a start codon (e.g., methionine (ATG)) and ends with a stop codon (e.g., TAA, TAG, or TGA), encoding a polypeptide. Heterogene-derived signal peptides are known in the art and can be incorporated into the nucleic acids of this disclosure after measuring their desired properties. In some embodiments, the signal peptide may include any of the following: MDSKGSSQKGSRLLLLLVVSNLLLPQGVVG (SEQ ID NO: 148), MDWTWILFLVAAATRVHS (SEQ ID NO: 149), METPAQLLFLLLLWLPDTTG (SEQ ID NO: 150), MLGSNSGQRVVFTILLVAPAYS (SEQ ID NO: 151), MKCLLYLAFLFIGVNCA (SEQ ID NO: 152), MWLVSLAIVTACAGA (SEQ ID NO: 153), and MFVFLVLLPLVSSQC (SEQ ID NO: 154). The nucleic acid molecules provided herein can be synthesized by solid-phase technology, liquid-phase chemistry, enzymatic ligation, or a combination thereof. The synthesized nucleic acid molecules may be purified and quantified. See specifically WO2021222304, the entire contents of which are incorporated herein by reference.

[0098] 7. Vector In another aspect, the Disclosure further provides a vector comprising a nucleic acid molecule provided herein, wherein the nucleic acid molecule is operably ligated to at least one polynucleotide regulator (e.g., a promoter) to express a chimeric protein construct encoded by the nucleic acid molecule. In some embodiments, the vector is selected from plasmids, nanoplasmides, cosmids, viral vectors, minicircles, RNA vectors, or linear or round DNA (e.g., transposon DNA) or RNA molecules. In some embodiments, the viral vector is selected from viral vectors selected from retroviruses, lentiviral vectors, adenoviruses, parvoviruses (e.g., adeno-associated viruses), adeno-associated virus (AAV) vectors, negative-strand RNA viruses such as coronaviruses, orthomyxoviruses (e.g., influenza virus), positive-strand RNA viruses such as rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., Machivirus and Sendai virus), picornaviruses and alphaviruses, and double-stranded DNA viruses, wherein the double-stranded DNA viruses include adenoviruses, herpesviruses (e.g., herpes simplex virus I and II, Epstein-Barr virus, cytomegalovirus), poxviruses (e.g., cowpox virus, fowlpox, and canarypox), Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, baculovirus, hepatitis virus, and virus-like particles (VLPs).

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

[0100] 8. Cell In another respect, the Disclosure further provides engineered cells that express the chimeric protein constructs provided in the Disclosure, or that contain the chimeric nucleic acid constructs or vectors provided in the Disclosure. In some embodiments, the engineered cells co-express the chimeric protein constructs provided in this disclosure and the co-expression moieties described above. The co-expression moieties and the target protein-binding domains of the chimeric protein constructs may be combined to achieve a particular purpose. For example, if the target protein-binding domain specifically recognizes a protein that can induce immune rejection expressed in immune cells, the co-expression moieties may be proteins that further suppress immune rejection (e.g., viral ER commensal glycoproteins), proteins that target the tumor microenvironment (e.g., chemokine receptors), or proteins that stimulate immune activity (e.g., cytokines, immune cell lineage proteins CD7, CD5, etc.), and the cells thus produced, when transplanted into the human body, have reduced immunogenicity (or immune rejection) and / or enhanced immune activity. This is highly useful for cellular immunotherapy and other therapeutic cell transplantation. Accordingly, the cells may be immune cells (e.g., T cells), stem cells, kidney cells, pancreatic islet cells, and cardiomyocytes, etc. In some embodiments, the cells are immune cells selected from T cells, natural killer (NK) cells, B cells, macrophages, monocytes, dendritic cells, neutrophils, or γδT cells. The T cells may be selected from a group of cells consisting of CD8+ T cells, CD4+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, natural killer T cells, γ-δT cells, cytokine-induced killer (CIK) T cells, tumor-infiltrating lymphocytes, and dominotoxic T cells.

[0101] In some embodiments, the engineered cells are T cells. When T cells with reduced immunogenicity and / or enhanced immune activity are obtained by co-expressing the chimeric protein construct described above, the T cells may further express receptors that specifically target tumor-related markers. These receptors include, but are not limited to, CARs, TCRs, and NK cell-activating receptor modules. In some embodiments, the engineered cells are T cells that co-express the chimeric protein construct provided in this disclosure with one or more selected from adenovirus E3-K19, CAR, NK cell activating receptor, and CNK signaling module. In some embodiments, the engineered cells are T cells that express together the chimeric protein construct provided in this disclosure, an NK cell activating receptor, and optionally a CNK signaling module. In some embodiments, the engineered cells are T cells that jointly express the chimeric protein construct, adenovirus E3-K19, and CAR provided in this disclosure. In another aspect, the disclosure further provides a method for producing engineered cells (e.g., T cells) as described herein, comprising introducing the vector described herein into starting cells under conditions suitable for expressing the nucleic acid molecules described herein.

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

[0103] Activation and / or proliferation of immune cells is one of the main steps of immune cell function. In some embodiments, immune cells are activated and proliferated before and / or after genetic modification. In some embodiments, immune cells are activated and / or proliferated in vitro, ex vivo, or in vivo. Methods for activating and proliferating immune cells are described in the art and may be used in the methods described herein. For example, T cells may be activated and proliferated by contacting a surface to which a drug that stimulates CD3 / TCR complex-related signaling and a ligand that stimulates costimulatory molecules on the T cell surface are attached. In particular, a T cell population can be stimulated by contact with, for example, an anti-CD3 antibody or its antigen-binding fragment or an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase C activator (e.g., briostatin) and a calcium ion carrier. To co-stimulate helper molecules on the T cell surface, ligands that bind to the helper molecules are used. For example, under conditions suitable for stimulating T cell proliferation, a T cell population may be contacted with anti-CD3 antibodies and anti-CD28 antibodies. Anti-CD3 antibodies and anti-CD28 antibodies can be used to stimulate the proliferation of CD4+ T cells or CD8+ T cells. In some embodiments, the primary and co-stimulatory signals for T cells may be provided by different protocols.

[0104] Genetically modified cells can be achieved by transduction of substantially homogeneous starting cells with nucleic acid molecules encoding the chimeric protein constructs provided herein. In some embodiments, retroviral vectors (e.g., lentiviral vectors) are used to introduce the nucleic acid molecules provided herein into starting cells. For example, the nucleic acid molecules provided herein may be cloned into a lentiviral vector and expression may be initiated from its endogenous promoter, lentiviral long-terminal repeat sequence, or a promoter specific to the target cell type of interest. Common delivery methods for viral vectors include, but are not limited to, electroporation, microinjection, gene guns, and magnetic transfection. Genetically modified cells can also be achieved by delivering nucleic acid molecules via LNPs. See, specifically, the contents of the “6. Nucleic Acid Molecules” subsection above. Other nonviral methods for delivering nucleic acids include in vitro transfection using calcium phosphate, DEAE glucan, electroporation, and protoplast fusion. Engineered cells described herein may also be obtained by achieving genetic modification of starting cells using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALE nucleases, CRISPR).

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

[0106] 9. Pharmaceutical compositions, kits In another aspect, this disclosure further provides (i) a chimeric protein construct, nucleic acid molecule, vector or cell population as described herein, and (ii) a pharmaceutically acceptable medium. The term “pharmaceutically acceptable medium” means any and all solvents, dispersions, coatings, antimicrobial / antifungal agents, isotonic agents and absorption retarders, etc., that can facilitate the storage and administration of the chimeric protein constructs, nucleic acid molecules, expression vectors and / or cell populations provided herein. A pharmaceutically acceptable medium may contain any suitable components, such as, but is not limited to, aqueous saline solutions, liposomes, polymerization excipients, colloids or vector particles. In some embodiments, the pharmaceutically acceptable medium is an aqueous salt solution capable of dissolving or dispersing the chimeric protein constructs, nucleic acid molecules, expression vectors, and / or cell populations provided herein. Specific examples of aqueous salt solutions include, but are not limited to, buffered salt solutions, sucrose solutions, physiological saline, acetate buffers, phosphate buffers, citrate buffers, bicarbonate buffers, salt solutions, and polysorbate solutions.

[0107] i) Nucleic acid drug compositions In some embodiments, the pharmaceutically acceptable medium is a liposome. A liposome is a monolayer or multilayer vesicle having a membrane formed of a lipophilic material and an internal aqueous portion. The nucleic acid molecules and / or vectors provided in this disclosure may be encapsulated in the aqueous portion of a liposome. Exemplary liposomes include, but are not limited to, those provided by 3[N-(N',N'-dimethylaminoethane)aminoformyl]cholesterol (DC-Chlo), liposomes provided by N-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA), and liposomes provided by 1,2-dioleoyloxy-3-trimethylethane (DOTAP). Methods for preparing liposomes and for encapsulating nucleic acid molecules and / or vectors in liposomes are well known in the art (see, for example, DD Lasic et al, Liposomes in gene delivery, published by CRC Press, 1997). In some embodiments, the pharmaceutically acceptable medium is a polymerization excipient, and is not limited to microspheres, microcapsules, polymer micelles, and dendritic polymers. The nucleic acid molecules and / or vectors provided herein can be encapsulated, attached to, or coated with polymer components by methods known in the art (see, for example, W. Heiser, Nonviral gene transfer techniques, published by Humana Press, 2004; U.S. Patent 6025337; Advanced Drug Delivery Reviews, 57(15): 2177-2202 (2005)).

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

[0109] The pharmaceutical compositions provided herein can be administered by any suitable route known in the art, including, but not limited to, extra-gastrointestinal, oral, intestinal, cheek, nose, topical, rectal, vaginal, intramuscular, intranasal, transmucosal, epidermal, transdermal, dermal, ocular, pulmonary, and subcutaneous administration routes. The pharmaceutical compositions provided herein can be administered to subjects in formulations or forms suitable for each administration route. Suitable formulations for administering the pharmaceutical compositions include, but are not limited to, solutions, dispersions, emulsions, powders, suspensions, aerosols, sprays, nasal drops, liposome formulations, patches, implants, and suppositories. The formulations exist in convenient unit dosage forms and can be prepared by any method known in the pharmaceutical field. Methods for preparing these formulations or pharmaceutical compositions include the step of providing the nucleic acid molecules described herein to one or more pharmaceutically acceptable media and optionally one or more excipients. Methods for producing such formulations can be found, for example, in Remington's Pharmaceutical Sciences (Remington: The Science and Practice of Pharmacy, 19th ed., AR 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); TW Kim et al., The Journal of Gene Medicine, 7(6): 749-758 (2005); and SF Jia et al., Clinical Cancer Research, 9:3462 (2003); A. Shahiwala et al., Recent patents on drug delivery and formulation, 1:1-9 (2007) (These references are incorporated herein by reference in their entirety) can be consulted.

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

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

[0112] Ionizable lipids can form lipid nanoparticle formulations together with cholesterol, auxiliary lipids, and polyethylene glycolated lipids (i.e., PEGylated lipids). Cholesterol is a naturally occurring rigid and hydrophobic lipid that can maintain the structure and stability of lipid nanoparticles. Cholesterol can also facilitate the fusion of mRNA-supported lipid nanoparticles (i.e., mRNA nanoparticles) with the endosomal membrane. Auxiliary lipids (e.g., the amphoteric lipids DOPE, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)) are widely used to facilitate cell membrane translocation and endosomal membrane escape (see, e.g., N. Chaudhary et al., Nat. Rev. Drug Discovery 2021, 20, 817). PEGylated lipids consist of PEG and an anchor lipid. The hydrophilic PEG is mainly distributed on the surface of the mRNA complex, while the hydrophobic region is embedded within the lipid bilayer. The introduction of PEGylated lipids not only extends the half-life of lipid nanoparticles but can also adjust particle size by altering 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 carbon atoms, respectively (see, for example, N. Chaudhary et al., Nat. Rev. Drug Discovery 2021, 20, 817).

[0113] Over the past several decades, researchers have studied DLin-MC3-DMA, SM-102, TT3, C12-200, and 306O. i10We have also developed large libraries of ionizable lipids for mRNA delivery, such as 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 these have yielded remarkable results in clinical applications. A typical example is DLin-MC3-DMA, a key component of Onpattro for siRNA delivery approved by the U.S. Food and Drug Administration (FDA) (see, e.g., A. Akinc et al., Nat. Nanotechnol. 2019, 14, 1084). DLin-MC3-DMA is also widely used for mRNA delivery, including protein and peptide substitution, gene editing, and antiviral infection (see, e.g., RS Riley et al., Sci. Adv. 2021, 7, eaba1028). Two "star molecules," SM-102 and ALC-0315, have been approved by the FDA as key components of the BNT162b and mRNA-1273 vaccines for preventing COVID-19, respectively (see, e.g., X. Hou et al., Nat. Rev. Mater. 2021, 10, 1078).

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

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

[0116] ii) Oncolytic viruses and their pharmaceutical compositions In some embodiments, the present application provides oncolytic viruses capable of expressing the chimeric protein constructs described herein. In this application, “oncolytic virus” means any virus capable of infecting tumor cells, replicating within tumor cells, and lysing tumor cells. In some embodiments, the oncolytic virus can further spread to other tumor cells in a continuous replication cycle. Oncolytic viruses may originate from multiple viruses, and non-specific examples include cowpox virus, adenovirus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), myxoma virus, reovirus, poliovirus, varicella stomatitis virus (VSV), measles virus (MV), lassa virus (LASV), Newcastle disease virus (NDV), and variants of these viruses. In some embodiments, the present application provides an oncolytic virus comprising a nucleic acid molecule provided herein, wherein the nucleic acid molecule is operably linked to at least one polynucleotide regulator (e.g., a promoter) to express a chimeric protein construct encoded by the nucleic acid molecule. In some embodiments, the oncolytic viruses described herein can infect substantially any cell type. In some embodiments, the oncolytic viruses may be replication-selective. Replication-selective oncolytic viruses replicate more in tumor cells than in non-tumor cells.

[0117] It should be understood that oncolytic viruses can be replication-selective if viral replication is under the control of gene expression regulators, such as the enhancer / promoter region derived from the 5' end of the albumin gene (see, e.g., Miyatake et al., 1997, J. Virol. 71:5124-5132). In some embodiments, oncolytic viruses may have tumor cell-specific promoters or tumor cell-specific transcriptional regulatory sequences in their genome. “Tumor cell-specific” means that, in the case of promoters or transcriptional term sequences, they are present in target tumor cells at levels typically higher than in normal cells. This allows for the conferring of enhanced tumor cell specificity to oncolytic viruses. For example, by operably linking the HSV gene to the TGF-β promoter, the main transcriptional units of HSV can be placed under the transcriptional regulation of the tumor growth factor-β (TGF-β) promoter. Certain tumor cells are known to overexpress TGF-β more than non-tumor cells of the same type. Therefore, oncolytic viruses whose replication is under the transcriptional regulation of the TGF-β promoter are replication-selective, as they replicate better in certain tumor cells than in non-tumor cells of the same type. Similar replication-selective oncolytic viruses can be constructed using any gene expression regulator known to selectively overexpress in affected cells. A replication-selective oncolytic virus may be, for example, an HSV-1 mutant in which the gene encoding ICP34.5 is mutated or deleted. Oncolytic viruses may also contain other modifications to their genome. For example, an oncolytic virus may contain additional DNA inserted into the UL44 gene. This insertion may cause functional inactivation of the UL44 gene and the resulting cleavage phenotype. Alternatively, this may be inserted into an already inactivated gene, or used to replace a deleted gene.

[0118] In some embodiments, the oncolytic virus is modified to place the nucleic acid sequence encoding the chimeric protein construct described herein under the control of a tumor cell-specific promoter. In some embodiments, the oncolytic virus is modified to place a gene encoding at least one protein necessary for viral replication under the control of a tumor cell-specific promoter. In some embodiments, the oncolytic viral vector may express the chimeric protein construct described herein. The targeting domain of the chimeric protein construct can recognize and bind to oncoproteins. Oncoproteins may 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 variants), or proteins that promote evasion of normal immune responses by tumor cells (e.g., PD-L1, TGF-β1).

[0119] In some embodiments, the targeting domain of the chimeric protein construct can bind to Bcl-2. Any targeting domain capable of targeting and binding to bcl-2 can be used. For example, a nanobody sequence for the BH1 and BH2 motifs, which are highly conservative and homologous to the Bcl-2 anti-apoptotic family member proteins, may be developed as a targeting domain. An ER-TPD element that targets Bcl-2 family proteins may be constructed and inserted behind a potent promoter of an oncolytic virus by cloning. After specifically infecting tumor cells, the oncolytic virus may proliferate in large quantities and express the ER-TPD element of the Bcl-2 family protein, thereby achieving degradation of the Bcl-2 anti-apoptotic member protein in the cell and directly inducing apoptosis in tumor cells. In some embodiments, the targeting domain of the chimeric protein construct can bind to VEGFR. Any targeting domain capable of targeting and binding to VEGFR can be used. For example, a recombinant scFv using the heavy and light chains of VEGFR antibodies (anti-VEGF-A antibody Bevacizumab and anti-VEGFR2 antibody Ramucizumab) is connected in series with an ER-TPD element to construct an ER-TPD element that targets VEGFR. This element is then inserted after a strong promoter of an oncolytic virus by cloning, and after specifically infecting tumor cells, the oncolytic virus proliferates rapidly and expresses the VEGFR ER-TPD element, directly inhibiting VEGF expression in tumor cells. As a result, the tumor cells lack sufficient nutrients, their growth is suppressed, and apoptosis is induced.

[0120] In some embodiments, the targeting domain of the chimeric protein construct can bind to TGF-β1 and PD-L1. Any targeting domain capable of targeting and binding to bcl-2 can be used. For example, a recombinant scFv using the heavy and light chains of TGF-β1 and PD-L1 antibodies may be connected in series with an ER-TPD element to construct an ER-TPD element that targets TGF-β1 and PD-L1. This element may then be inserted behind a potent promoter of an oncolytic virus by cloning, and after specifically infecting tumor cells, the oncolytic virus may proliferate rapidly and express the TGF-β1 and PD-L1 ER-TPD element, directly inhibiting and degrading the expression of TGF-β1 and PD-L1 within tumor cells, disrupting the tumor's immunosuppressive microenvironment, and achieving a therapeutic effect on the tumor. While not limited by theory, the chimeric protein constructs described in this application are thought to have unique advantages when expressed in tumor cells by oncolytic viral vectors. These chimeric protein constructs can promote apoptosis or cell death in tumor cells by targeting and degrading proteins that aid in tumor cell growth or immune evasion, or that resist apoptosis. In addition, oncolytic viral vectors can specifically replicate within tumor cells and promote tumor cell lysis through viral replication and degradation. By incorporating two different tumor-killing mechanisms into an oncolytic viral vector, a synergistic killing effect can be achieved in a single pharmaceutical form.

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

[0122] iii) Cell therapy compositions In another aspect, the present disclosure further provides cell therapy compositions comprising engineered cells or cell populations and pharmaceutically acceptable media provided herein. Exemplary media include buffers such as neutral buffered salt aqueous solutions and phosphate buffered salt aqueous solutions; carbohydrates such as glucose, mannose, sucrose or glucan, and mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In one aspect, the cell therapy compositions of the present invention are prepared for intravenous administration.

[0123] III. Applications of TPD In some respects, this disclosure further provides applications in the preparation of therapeutic cells for the chimeric protein constructs, nucleic acid molecules, or vectors described herein. In some respects, this disclosure further provides applications in the treatment of diseases for the chimeric protein constructs, nucleic acid molecules, or vectors described herein. In some respects, this disclosure further provides applications in the treatment of diseases for cells expressing the chimeric protein constructs, nucleic acid molecules, or vectors described herein.

[0124] 1. Cell transplantation In one aspect, the present disclosure further provides uses of the chimeric protein constructs, nucleic acid molecules or vectors described herein in the production of cells to be transplanted (e.g., allogeneic cell transplantation). In some 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, and the like. In some embodiments, the cells to be transplanted may be autologous cells or allogeneic cells. In one aspect, the present application further provides cells to be transplanted that express the chimeric protein constructs, nucleic acid molecules or vectors described above. Herein, the cells express the chimeric protein construct described in the present application, the chimeric protein construct comprises an ERAD machinery protein binding domain and a targeting domain, the targeting domain comprises a domain that specifically targets or binds a target protein, and the target protein is a transplantation rejection-related protein. In some embodiments, the chimeric protein construct further comprises a proteolytic pathway member (e.g., E3 ubiquitin ligase, proteasome, lysosome) binding domain as described in the present application. Any of the proteolytic pathway member binding domains described in the present application can be used.

[0125] In some embodiments, the transplantation rejection-related proteins include antigen-presenting molecules (e.g., MHC class I molecules, MHC class II molecules, MICA / B molecules, etc.), antigen recognition molecules (e.g., TCR, CD123, NKG2D, etc.), immune checkpoint molecules (e.g., PD-1, PD-L1, CTLA4, TIM3, TIGIT, LAG3, A2AR, BTLA, IDO1, IDO2, TDO, KIR, NOX2, VISTA, SIGLEC7, PVR, etc.), and the like. In some embodiments, the transplantation rejection-related proteins include immunogenic proteins (e.g., HLA (HLA α / β), TCR (αβ TCR), NKG2D (natural killer cell group 2 member D) ligands, etc.). The present disclosure further provides the aforementioned transplanted cells and the therapeutic method thereof, which can overcome the problems in conventional cell transplantation and cell therapy, such as the source of individualized transplanted cells and therapeutic cells, the impossibility of standardized and scaled-up production, and the reduction of therapeutic efficiency.

[0126] The immune system is highly plastic and has an almost infinite ability to detect invading viruses, bacteria, exogenous cells and lesion cells. This remarkable capability in immunological surveillance is mainly achieved by humoral immunity and cell-mediated immunity, involving two important molecular structures: immunoglobulins and T cell receptors (TCRs). A TCR is the defining structure of T cells, which is a transmembrane heterodimer consisting of α and β chains or δ and γ chains linked by disulfide bonds. In these chains, the complementarity-determining regions (CDRs) determine the antigen to which the TCR binds. In a TCR, the TCRα and TCRβ subunits (or TCRγ and TCRδ in γδ T cells) are responsible for recognizing the major histocompatibility complex (MHC) / antigen ligand. The human MHC class I gene region contains alleles at the HLA-A, B, C loci, and encodes typical class I antigens (molecules) such as HLA-A antigen, B antigen and C antigen, which are referred to 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. When cells produce mutant proteins, or foreign bacteria or viruses invade, the cells present these mutant protein or heterologous protein antigen epitopes, so that immune cells recognize them and carry out immune attack and killing, thereby removing lesion cells, pathogenic bacteria and viruses that have invaded the cells. α / β T lymphocytes recognize peptide-MHC ligands via 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 to the antigen and three invariant dimers (CD3γε, δε, and ζζ) involved in TCR.CD3 surface transport, stabilization, and signaling. The αβ T cell receptor (αβ TCR) is expressed in most T cells (approximately 95%) and plays a crucial 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) in allogeneic hematopoietic stem cell transplantation (allo-HCT) and allogeneic CAR-T cell therapy.

[0127] The human leukocyte antigen (HLA) system or complex is a group of related proteins encoded by the human major histocompatibility complex (MHC) gene complex. These cell surface proteins are involved in regulating the immune system. In a therapeutic transplantation setting, an "HLA mismatch" occurs when the donor HLA on an allogeneic graft differs from that of the recipient. HLA mismatch can lead to the activation of allogeneic reactive T cells and cause acute cell rejection (ACR) within 6 months post-transplantation. Mismatched donor HLA antigens are also targets for the development of novel donor-specific HLA antibodies (dnDSA), which exert an enhancing effect in acute and chronic transplant cell (e.g., T cell) rejection. Therefore, it is recommended to effectively block graft-versus-host disease (GVHD) by genetically disrupting the TCR to generate safe allogeneic infusion and versatile transplant cells (e.g., T cells) for therapeutic purposes. Furthermore, it is necessary to inhibit HLA expression in allogeneic cells (e.g., T cells) to reduce the recipient immune system's rejection response to allogeneic T cells (TCRαβ and / or HLA class I). In some embodiments, transplanted cells expressing the above-described chimeric protein constructs, nucleic acid molecules, or vectors have reduced immunogenicity.

[0128] In some embodiments, the transplanted cells having reduced immunogenicity (or high compatibility) as described herein are T cells, and the TPD chimeric protein construct expressed therein comprises the transmembrane domains or functional variants of scFv and HCMV glycoproteins US2 and / or US11 that specifically recognize TCR and / or HLA, as well as the endoplasmic reticulum commensal domain or functional variants thereof. The ERAD mechanism protein-binding domain includes the amino acid sequence shown in SEQ ID NO: 76 or SEQ ID NO: 82, or an amino acid sequence having at least 80% (for example, at least 85%, at least 90%, at least 95%, or at least 99%) identity to these. In some embodiments, the transplanted cells having reduced immunogenicity are T cells, and the ER-TPD chimeric protein construct expressed therein includes a transmembrane domain or functional variant of scFv and adenovirus E3-K19 that specifically recognize the TCR, as well as an endoplasmic reticulum (ER) resident domain or functional variant thereof. In some embodiments, the transplanted cells having reduced immunogenicity are T cells, and they express an ER-TPD chimeric protein construct comprising a target protein-binding domain and an ERAD mechanism protein-binding domain, wherein the target protein-binding domain comprises six CDRs contained in the amino acid sequence 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 the amino acid sequence 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.

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

[0130] In another aspect, the present disclosure provides a method for reducing a subject's immune response to cell transplantation. This method reduces the subject's immune response to the graft or the modified cells by administering to the subject a therapeutically effective amount of the transplanted cells having the reduced immunogenicity. In some embodiments, the transplanted cells express a TPD chimeric protein construct described herein intracellularly, the TPD chimeric protein construct comprising a domain that targets immunogenic proteins (e.g., HLA (HLAα / β), TCR (αβTCR), NKG2D (natural killer cell family 2 member D) ligand, etc.), the ERAD mechanism protein-binding domain described above, and optionally a proteolytic pathway member-binding domain.

[0131] 2. Cell therapy Cell therapy involves modifying cells (e.g., immune cells) using genetic engineering techniques to make them present tumor-associated antigens or express receptors that specifically recognize diseased cells. These cells are then exponentially proliferated in vitro and injected into the patient's body to activate the immune system, attacking tumor cells or directly and specifically recognizing and killing diseased cells. In 2012, CD19-targeting CAR-T cells achieved the first-ever targeted removal of tumor cells in B-cell leukemia patients in the history of human medicine. This became another treatment technology capable of completely curing leukemia, following bone marrow stem cell transplantation, and ushered in a new era of precision medicine cell therapy. This technology is expected to have applications in the treatment of various hematological malignancies and solid tumors. However, the clinical efficacy of conventional CAR-T therapy for solid tumors has been poor, for the following reasons. (1) The killing function of CAR-T depends heavily on the recognition of tumor-associated antigens (TAAs) by the CAR structure, but due to the heterogeneity of solid tumors, there are significant differences in the expression of target proteins on the surface of tumor cells; conventional single-target CAR-Ts cannot completely recognize and kill all tumor cells, leading to immune evasion and recurrence / metastasis of tumors; (2) The direct immunosuppressive effect of solid tumors, such as the expression of PD-L1 and B3H7, directly inhibits T cell activation; (3) The immunosuppressive microenvironment of solid tumors contains many immunosuppressive cells such as TAMs, Tregs, and MDSCs, making it difficult for conventional CAR-T designs to overcome the constraints imposed by immunosuppressive cells within the tumor; (4) Conventional CAR-Ts are individualized cell therapies, and the T cell source is the patient themselves. If a patient receives many radiotherapy or chemotherapy treatments, the function of the immune system is impaired, making it impossible to isolate a sufficient amount of T cells from the patient's peripheral blood. Even if isolated and modified, the proliferation and killing functions of the T cells are weak, making it difficult to exert a therapeutic effect. The transplantable cells (e.g., T cells) provided in this invention can overcome the problems faced by conventional cell therapies, such as differences in tumor targets, the inhibitory environment for tumor immunity, and the cell source for personalized therapy.

[0132] For the purpose of cell therapy, transplanted cells with reduced immunogenicity (e.g., T cells, and even general-purpose T cells with impaired TCRαβ and MHC molecular surface expression) may be genetically modified. For example, the therapeutic objective may be achieved by expressing the co-expression region described above. For example, to enable transplanted cells (e.g., T cells) to specifically act on disease-related target proteins or cells expressing disease-related target proteins, transplanted cells (e.g., T cells) having reduced immunogenicity may express a binding domain or molecule that recognizes and binds to disease-related target proteins, such as a chimeric antigen receptor (CAR), an engineered TCR, the CNK receptor provided in this application, or an antiviral protein binding domain. In some embodiments, the transplanted cells (e.g., T cells) having reduced immunogenicity may express molecules (e.g., chemokine receptors or cytokine receptors) that can mediate the movement of the transplanted disease into the disease microenvironment. Furthermore, for example, an immunostimulatory molecule may be expressed in the transplanted cells (e.g., T cells) having reduced immunogenicity so that the transplanted cells (e.g., T cells) have additional immunostimulatory activity.

[0133] In another respect, the present invention further provides that by introducing NK elements, particularly optimized recombinant NK elements, into transplantable cells (e.g., UT cells) provided in the present invention, T cells can effectively and broadly recognize all virus-infected cells and tumor cells, similar to NK cells, and optionally, by further optimizing the signaling elements, CNK-T cells can effectively activate and kill tumor cells. Since NK targets include family member proteins such as MICA, MICB, and ULBP1-6, which are widely expressed in various tumor cells and cover each stage of tumor progression, CNK technology can effectively resolve off-target effects by single CAR-T cells and eliminate the possibility of tumor immune evasion. In addition, the chimeric adapter introduced into the design of CNK can effectively transmit and amplify NK signals, overcome the constraints of immune checkpoints such as PD1 signaling, effectively activate T cells, and achieve tumor cell killing. Chimeric adapters can amplify CNK-T cell signaling, resist immunosuppression in the tumor environment, achieve T cell activation, and kill tumor cells; CNK-T cells can also eliminate immunosuppressive cells such as MDSCs by recognizing targets via NK. After infecting cells, viruses express specific functional proteins that inhibit the presentation of viral antigen epitopes and induce immune evasion by directly promoting the assembly and transport of MHCIs or the targeted degradation of MHCI molecules.

[0134] In some respects, this disclosure provides UT cells that further express the CNK receptor and optionally CAR. Such UT cells are also called "CNKT-UT". In this application, it has been verified that CNK-UT has broad-spectrum tumor recognition and killing ability. In this application, it has been further verified that CAR / CNK-UT products are tailored to specific targets and that CAR / CNK-UT products have more potent killing and activating capabilities against tumor cells compared to conventional CAR-T; in animal experiments, CAR / CNK-UT products also have more effective tumor removal capabilities. UT technology achieves isogeneic modification, and since T cells are provided from healthy donors, it enables standardization and scale-up production of CNK-UT products, allowing them to be pre-fabricated and ensuring the killing and activating capabilities of T cells against tumor cells. In some preferred embodiments, the CNKT-UT comprises a nucleic acid molecule encoding an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 117, preferably 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity. In some preferred embodiments, the CNKT-UT comprises a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO: 117. In another aspect, the present disclosure further provides applications of CNKT-UT in the manufacture of pharmaceuticals for the treatment of diseases.

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

[0136] While not limited by theory, certain target proteins are expressed in both target cells (e.g., cancer cells or virus-infected cells) and therapeutic immune cells. Typically, such targets are difficult to treat with cell therapy. Therefore, when therapeutic immune cells express CARs that target such targets, the CARs also recognize the same targets expressed by the immune cells themselves. This leads to autophagy in the immune cells, preventing normal proliferation and limiting the killing effect on target cells. The chimeric protein construct provided in this application can degrade the target protein expressed by the immune cells themselves, thereby avoiding autophagy while maintaining a specific killing effect on target cells. Another advantage of using the chimeric protein construct according to this application is that co-expression allows for the simultaneous expression of the chimeric protein construct that targets this target and the binding domain (e.g., CAR, TCR, etc.) that targets this target. This is simpler and more effective than conventional methods such as gene knockout. In some embodiments, the chimeric protein construct further includes a proteolytic pathway member (e.g., E3 ubiquitin ligase, proteasome, lysosome) binding domain described in this application. Any of the proteolytic pathway member binding domains described in this application may be used.

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

[0138] In some embodiments, the dose of total cells and / or the dose of individual subgroups of cells is 10 4 Individual cells or about 10 4 Individual cells / kilogram (kg) body weight ~10 9 Individual cells or about 109 cells per kilogram (kg) of body weight, for example, 10 5 to 10 6 cells per kg of body weight, for example, at least or at least about 1×10 5 cells / kg, 1.5×10 5 cells / kg, 2×10 5 cells / kg or 1×10 6 cells / kg of body weight, alternatively 1×10 5 cells / kg, 1.5×10 5 cells / kg, 2×10 5 cells / kg or 1×10 6 cells / kg of body weight or about 1×10 5 cells / kg, 1.5×10 5 cells / kg, 2×10 5 cells / kg or 1×10 6 cells / kg of body weight. For example, in some embodiments, the cells are administered at 10 4 T cells or about 10 4 T cells per kilogram (kg) of body weight to 10 9 T cells or about 10 9 T cells per kilogram (kg) of body weight, or within a certain error range thereof, for example, 10 5 to 10 6 T cells per kg of body weight, for example, administered at least or at least 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 of body weight, alternatively 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 of body weight 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 of body weight.

[0139] Cells can be administered by any suitable means. For example, they can be delivered by bolus, intravenous or subcutaneous injection, intraocular injection, periorbital injection, subretinal injection, intravitreous injection, transseptal injection, subscleral injection, choroidal injection, precautionary injection, subperineal injection, subconjunctival injection, sub-Tenon injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral injection. In some embodiments, they are administered extra-gastrointestinal, intrapulmonary, and intranasal, and intra-lesional if local treatment is desired. Extra-gastrointestinal infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. In some embodiments, a prescribed dose of cells is administered by a single bolus. In some embodiments, cells are administered by several boluses, for example, within 3 days, or by continuous infusions. Some embodiments provide a repeated dosing method in which a first dose of cells is administered, followed by one or more second consecutive doses. When administered to subjects in adoptive therapy, the timing and size of multiple cell doses are typically designed to increase the efficacy and / or activity and / or function of antigen-expressing T cells (e.g., CAR-expressing T cells). In some embodiments, repeated dosing reduces the downregulation or inhibitory activity that may occur when inhibitory immune molecules such as PD-1 and / or PD-L1 are upregulated in antigen-expressing T cells such as CARs. Such methods include administering a first dose, usually followed by one or more consecutive doses, with a specific time range between each dose.

[0140] In the context of adoptive cell therapy, administration of a given "dose" includes administration of a given amount or number of cells as a single composition and / or a single continuous administration (e.g., as a single injection or continuous infusion), and also includes administration in which the dose of the given amount or number of cells provided by multiple individual compositions or infusions over a predetermined period (within 3 days) is a divided dose. Accordingly, in some cases, the first or consecutive dose is a single or consecutive administration of a specific number of cells that is administered or initiated at a single point in time. However, in some cases, the first or consecutive dose is administered over a period of no more than 3 days, for example, by injection or infusion several times once every 3 days or 2 days or 1 day, or by infusion several times a day.

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

[0142] In some embodiments, the method includes introducing a nucleic acid molecule (e.g., mRNA) or vector (e.g., a viral vector) encoding the chimeric protein construct described herein into an individual requiring such that the nucleic acid molecule or vector expresses the chimeric protein construct in the cells of the individual. Any conventional route of administration, such as a non-enteral route, a local route, or a mucosal route, may be used in the present invention. Non-enteral routes include injection or intravenous administration and include systemic and local routes. Generally, types of non-enteral injections include intravenous (into a vein), intra-arterial (into an artery), intracutaneous (into the dermis), subcutaneous (below the epidermis), muscle (into the muscle), and intratumoral (into the tumor or a site very close to the tumor). Typical intravenous administration is via a venous route. Mucosal administration includes, but is not limited to, routes of oral / esophageal, nasal, tracheal, intrapulmonary, vaginal, or rectal administration. Local administration can also be performed by percutaneous means (e.g., a patch). For administration, conventional syringes and needles, or any known compound or device that can facilitate or improve the delivery of the active agent to the target in the prior art, may be used. Depending on the disease to be treated in the individual and the pathogenic protein to be degraded, an appropriate chimeric protein construct, as well as its nucleic acid molecule and vector, may be selected. In some preferred embodiments, the disease includes various solid tumors, hematological malignancies, infectious diseases (e.g., viral infections), autoimmune diseases, neurodegenerative diseases, and metabolic diseases.

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

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

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

[0146] In some preferred embodiments, gastrointestinal viral diseases include polio; Cooksackievirus infection; ECHO virus infection; and viral gastroenteritis includes rotavirus gastroenteritis, Norwalk virus gastroenteritis, adenovirus gastroenteritis, astrovirus gastroenteritis, coronavirus gastroenteritis, and calicivirus gastroenteritis, etc. In some preferred embodiments, liver viral diseases include viral hepatitis A, viral hepatitis B, viral hepatitis C, viral hepatitis D, viral hepatitis E, EB virus hepatitis, and cytomegalovirus hepatitis, among others. In some preferred embodiments, skin and mucous membrane viral diseases include measles, rubella, infantile acute rash, varicella-zoster, smallpox, herpes simplex virus infection, rabies, and foot-and-mouth disease. In some preferred embodiments, the ocular viral disease includes epidemic keratoconjunctivitis, follicular conjunctivitis, and herpetic keratoconjunctivitis, among others. In some preferred embodiments, central nervous system viral diseases include epidemic B encephalitis, Western equine encephalitis, Eastern equine encephalitis, St. Louis encephalitis, Venezuelan equine encephalitis, Murray Valley encephalitis, California encephalitis, forest encephalitis, and lymphocytic choroid plexus meningitis. In some preferred embodiments, lymphocytic viral diseases include infectious mononucleosis, cytomegalovirus infection, and acquired immunodeficiency syndrome.

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

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

[0149] Example 1: Design of ER-TPD chimeric protein constructs In this example, five chimeric protein constructs were designed using endoplasmic reticulum-based targeted protein degradation (ER-TPD). Of these, the first structure is a basic ER-TPD chimeric protein construct, while the other four structures are more potent ER-TPD chimeric protein constructs formed by adding an E3 ubiquitin ligase ligand, an E2 ubiquitin-conjugating enzyme ligand, or a lysosomal ligand, or a combination thereof, to the first structure. The ER-TPD chimeric protein construct contains an integrated multifunctional domain or a functional variant of a recombinant protein molecule that binds to a target protein and causes its targeted degradation.

[0150] 1.1 Basic ER-TPD Chimeric Protein Constructs In some embodiments, a basic ER-TPD chimeric protein construct TBD-TMD-ERD is used. Its linear schematic and structural schematic are shown in Figure 1A. It comprises three domains: (1) a targeted protein binding domain (TBD) or a functional variant thereof, which may be a target affinity ligand protein molecule or a functional variant thereof (which may be an antibody fragment, polypeptide, native receptor, ligand, or a recombinant protein having affinity for the target protein or its binding domain); (2) a transmembrane domain (TMD) or a functional variant thereof of a viral endoplasmic reticulum (ER) commensal glycoprotein; and (3) an ER retention domain (ERD) or a functional variant thereof of a viral endoplasmic reticulum commensal glycoprotein. The targeted domain or its functional variant, the transmembrane domain or a functional variant thereof of a viral endoplasmic reticulum commensal glycoprotein, and the cytoplasmic domain or a functional variant thereof of a viral endoplasmic reticulum commensal glycoprotein may be linked by hinges or linkers.

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

[0152] 1.2 ER-TPD chimeric protein constructs having an E3 ubiquitin ligase ligand domain In some embodiments, an ER-TPD chimeric protein construct TBD-TMD-ERD-E3L having an E3 ubiquitin ligase ligand domain is used. Its linear schematic and structural schematic are shown in Figures 2A and 2B, respectively. It comprises four domains: (1) a targeting domain or a functional variant thereof, which may be a target affinity ligand protein molecule or a functional variant thereof (which may be an antibody fragment, polypeptide, native receptor, ligand, or artificial recombinant protein domain having affinity for a target protein); (2) a transmembrane domain or a functional variant thereof of a viral endoplasmic reticulum commensal glycoprotein; (3) a cytoplasmic domain or a functional variant thereof of a viral endoplasmic reticulum commensal glycoprotein; and (4) an E3 ubiquitin ligase ligand (ligand for E3 ligase, E3L) or a functional variant thereof, which may be a polypeptide or recombinant protein and bind to numerous extraendoplasmic reticulum E3 ubiquitin ligases. The targeting domain or its functional variant, the transmembrane domain of the viral endoplasmic reticulum (ER) commensal glycoprotein or its functional variant, the cytoplasmic domain of the viral ER commensal glycoprotein or its functional variant, and the E3 ubiquitin ligase ligand domain or its functional variant may be linked by hinges or linkers. Since the number of ER-specific E3 ubiquitin ligases is limited, and the ERAD pathway for misfolded proteins within the ER competes for E3 ubiquitin ligases, effective degradation of target proteins cannot be achieved unless the extra-ER ubiquitination and proteasome degradation pathways are increased. Therefore, the inventors have found that effective ubiquitination and degradation of target proteins can be achieved by exogenously increasing the E3 ubiquitin ligase ligand.

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

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

[0155] As shown in Figure 3C, the mechanism of action of the ER-TPD chimeric protein construct TBD-TMD-ERD-E2L is as follows: [1] Specific binding and retention: After expression, the chimeric protein construct TBD-TMD-ERD-E2L resides in the endoplasmic reticulum and binds to the target protein via its targeting domain, retaining the target protein within the endoplasmic reticulum and preventing the transport of the synthesized target protein to the Golgi apparatus. [2] Reverse transport: After the chimeric protein construct binds to the target protein, the complex formed from the chimeric protein construct and the target protein is transported from the endoplasmic reticulum to the cytoplasm by the reverse transporter in the ERAD mechanism. Typical reverse transporters include Derlin, the E3 ligase Hrd1 and its chaperonin Sel1L, and can recruit cytoplasmic ATPase p97. Derlin has six transmembrane domains, both of which are located in the cytoplasm, forming transport channels, and the reverse transport process requires the involvement of ATPase p97. [3] Ubiquitination and proteasome degradation: The chimeric protein construct binds directly to the E2 ubiquitase via its E2 ubiquitase ligand domain, and the target protein is ubiquitinated by the E2 ubiquitase and transported to the proteasome for degradation. The E2 ubiquitase ligand domain can achieve ubiquitination and proteasome degradation of the target protein by binding to the UBC structure of the E2 ubiquitase via a RING zinc finger domain derived from an IAP family protein. Here, the ligand targeting the E2 ubiquitase may be a RING zinc finger domain derived from an IAP family protein or a recombinant protein sequence having a RING zinc finger domain. IAPs (inhibitors of apoptosis proteins) are a family of apoptosis protein inhibitors.IAPs can inhibit cellular apoptosis by acting as E3 ligases for pro-apoptotic proteins (e.g., caspases, SMACs, ARTS), achieving ubiquitination and degradation of these proteins; and they can also modulate NOD signaling and immune responses by ubiquitinating RIPK2 (receptor-interacting serine / threonine-protein kinase 2). Five members of the IAP family (XIAP, cIAP1, cIAP2, Livin, and ILP2) all possess a RING zinc finger domain and can bind to ubiquitinating enzyme E2 and the Apollon protein (Mikihiko Naito et al. SNIPERs-Hijacking IAP activity to induce protein degradation. Drug Discov Today Technol. 2019 Apr;31:35-42). Therefore, by linking the chimeric protein construct with the ubiquitosis-conjugating enzyme E2 via the RING zinc finger domain derived from an IAP family member, more effective ubiquitination and proteasome-mediated degradation of the target protein can be achieved. The following sequences can be used to design the E2 ligand.

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

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

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

[0159] Chaperone-mediated autophagy (CMA) is a process of selective protein autophagy and lysosomal degradation. The chaperone Hsc70 recognizes and targets a KFERQ-like motif on a target protein, and also recognizes the receptor protein LAMP2A on the lysosomal membrane, thereby transporting the target protein to the lysosome for degradation. Here, the KFERQ-like motifs are: KFERQ derived from RNase A (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); and 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) derived from aldolase B; EFLKQ (SEQ ID NO: 130) derived from Annexin I; QKVFD (SEQ ID NO: 131) derived from Annexin II; QELLR (SEQ ID NO: 132) derived from Annexin IV; Annexin QEFIK (SEQ ID NO: 133) derived from VI; RKVEQ (SEQ ID NO: 134) derived from aspartate aminotransferase; NLLKE derived from c-fos; NRVVD (SEQ ID NO: 135) derived from GAPDH; NKKFE (SEQ ID NO: 136) derived from glutathione transferase; VKELQ (SEQ ID NO: 137) derived from IκB; VDKLN (SEQ ID NO: 138) and RIKEN (SEQ ID NO: 139) derived from α-2-microglobulin; DVVRQ (SEQ ID NO: 140), QRIVE (SEQ ID NO: 141), and QLLRE (SEQ ID NO: 142) derived from Pax-2; IEKLQ (SEQ ID NO: 143) derived from 26S proteasome (C8); QEKVF (SEQ ID NO: 144) derived from 19S proteasome (PA28); QDLKF (SEQ ID NO: 145) derived from pyruvate kinase; VKKDQ (SEQ ID NO: 146) derived from α-synuclein (Massey AC. (et al. Chaperone-Mediated Autophagy in Aging and Disease. Current Topics in Developmental Biology, 2006; 73:205-235) This is a possible possibility.

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

[0161] As shown in Figure 5C, the mechanism of action of the ER-TPD chimeric protein construct TBD-TMD-ERD-E3L-LL is as follows: [1] Specific binding and retention: After expression, the chimeric protein construct TBD-TMD-ERD-E3L-LL resides in the endoplasmic reticulum and binds to the target protein via its targeting domain, retaining the target protein within the endoplasmic reticulum and preventing the transport of the synthesized target protein to the Golgi apparatus. [2] Reverse transport: After the chimeric protein construct binds to the target protein, the complex formed from the chimeric protein construct and the target protein is transported from the endoplasmic reticulum to the cytoplasm by the reverse transporter in the ERAD mechanism. [3] Lysosomal endocytosis and enzymatic degradation and ubiquitination / proteasome degradation: The chimeric protein construct is transported outside the endoplasmic reticulum, directly binds to lysosomal chaperones via lysosomal ligand domains, and is also transported to lysosomes via chaperones, where the target protein is degraded in lysosomes by various hydrolytic enzymes; a portion of the chimeric protein is transported outside the endoplasmic reticulum; the E3 ligase ligand in the chimeric protein construct can bind to the E3 ligase complex, where the target protein is ubiquitinated by E2 ubiquitin-conjugating enzymes in the complex and transported to the proteasome for degradation.

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

[0163] Example 3: Production of an expression plasmid for an ER-TPD chimeric protein construct that targets the TCR. The coating nucleotide sequences for the ER-TPD chimeric protein constructs TCR-ER-TPD (TCR-TPD1 and TCR-TPD2), which target the TCR designed in Example 2, were synthesized by whole-gene synthesis. Here, the nucleotide sequence of TCR-TPD1 is shown in SEQ ID NO: 160, and the protein it encodes is shown in SEQ ID NO: 161; the nucleotide sequence of TCR-TPD2 is shown in SEQ ID NO: 162, and the protein it encodes is shown in SEQ ID NO: 163. By molecular cloning, the nucleotide sequences of TCR-TPD1 and TCR-TPD2 were inserted into the lentiviral vector pCDH-CMV-MCS-EF1α-Puro (purchased from Youbio), respectively, to construct the TCR-ER-TPD lentiviral target vector. After confirming the accuracy by sequencing, DH5α-competent cells (Thermo Fisher) were transformed with the plasmids, single colonies were collected, and after large-scale culture, the plasmids were extracted using the PureLink plasmid extraction kit. TM Plasmid production and purification were performed using the HiPure Plasmid Maxiprep Kit (Thermo Fisher) to obtain TCR-ER-TPD lentivirus plasmids, namely TCR-TPD1 lentivirus plasmid and TCR-TPD2 lentivirus plasmid, respectively.

[0164] Example 4: Virus production of ER-TPD chimeric protein constructs that target the TCR The TCR-ER-TPD lentiviral plasmid obtained in Example 3 with a ratio of 1.64 pmol:1.3 pmol:0.72 pmol, along with the packaging plasmids psPAX2 and pMD2.G (purchased from Addgene, catalog numbers: 12259 & 12260), were co-transfected into 293 T cells (ATCC product, product number CRL3216TM) using polyethyleneimine transfection reagent (408727, Sigma) at a ratio of DNA:μg PEI = 1:3. For specific instructions on how to prepare the packaging plasmid, see PureLink.TM Refer to the instructions for the HiPure Plasmid Maxiprep Kit (K210006, Thermo). For specific transfection procedures, refer to the Sigma transfection instructions. After 16 hours of transfection, the culture medium was replaced with complete medium (purchased from Life Technologies, product code: 11995-065), and after incubation for 24, 48, and 72 hours, the lentivirus supernatant was collected. The combined supernatant was centrifuged at -80°C and 3000 rpm for 10-15 minutes, filtered through a 0.45 μm filter, and finally, the virus was concentrated by ultracentrifugation at 4°C and 25000 rpm for 2-3 hours. The concentrated TCR-ER-TPD lentivirus (TCR-TPD1 lentivirus or TCR-TPD2 lentivirus) was collected and stored at -80°C.

[0165] Example 5: Production of TCR ER-TPD Jurkat cells Thawed Jurkat cells were cultured in RMPI1640 medium supplemented with 5% FBS in an incubator at 37°C and 5% CO2. The medium was changed every 2-3 days, and the cell density was increased to 1 × 10⁶. 6 Cells were maintained at a single-cell / ml concentration. After the cell state recovered, the recovered cells were transfected with a lentivirus carrying the TCR-ER-TPD lentivirus plasmid prepared in Example 4. The specific procedure is as follows: Concentration of 0.5 × 10⁶ 6Jurkat cells were diluted to a single cell / ml concentration and spread in a 24-well plate. The TCR-TPD1 lentivirus and TCR-TPD2 lentivirus concentrates prepared in Example 4 were taken and added to the cells to achieve an MOI of 1. Polybrene 5 μg / ml was added, and the cells were placed in a flat centrifuge and centrifuged at low speed (500 g to 1000 g / min) for 30 to 60 minutes. Subsequently, the cells were cultured in an incubator at 37°C, and after 48 hours of transfection, Jurkat cells expressing TCR-ER-TPD (TCR ER-TPD Jurkat cells) were obtained. Cell phenotype detection was then performed by flow cytometry. After continuing the culture for 96 hours and 6 days, the following phenotypic detection and cell function experiments were performed.

[0166] Example 6: Phenotypic detection of ER-TPD transfected cells Flow cytometry was performed on the TCR ER-TPD Jurkat cells prepared in Example 5. Figure 7 shows the cell phenotype detection results for TCR ER-TPD Jurkat cells at 48 h (A), 96 h (B), and day 6 (C). In cell culture and flow cytometry, 5 × 10⁻¹⁶ cells were detected. 5Untransfected Jurkat cells and Jurkat cells transfected with the TCR-ER-TPD lentiviral vector were cultured separately. The cells were stained with the antibodies CD8-Pacific Blue, CD4-APC, EGFR-PE, and TCR-αβ-APC-Cy7 (all purchased from Biolegend), and cell phenotyping was performed using a flow cytometer (BD FACSCanto II). The following findings were obtained: (A) After 48 hours of culture, untransfected Jurkat cells expressed ...

Claims

1. A chimeric protein construct for degrading a target protein, The aforementioned chimeric protein construct comprises an endoplasmic reticulum-associated degradation (ERAD) mechanism protein-binding domain, a targeting domain, and a protein degradation pathway member-binding domain. The ERAD mechanism protein-binding domain comprises the transmembrane domain of the viral endoplasmic reticulum (ER) resident protein and the cytoplasmic domain of the viral endoplasmic reticulum (ER) resident protein. The protein degradation pathway member binding domain is linked to the ERAD mechanism protein binding domain, A chimeric protein construct selected from the group consisting of an E3 ubiquitin ligase ligand, a proteasome ligand, and a lysosomal ligand, wherein the protein degradation pathway member binding domain is selected.

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

3. The chimeric protein construct according to claim 1, wherein the viral endoplasmic reticulum commensal protein is not adenovirus E3-19K.

4. The chimeric protein construct according to claim 3, wherein the viral endoplasmic reticulum commensal protein is at least one selected from the group consisting of HCMV glycoproteins US2, US11, US3, US10, US6, HSV ICP47, CPXV12, BHV UL49.5, EBV BNFL2a, HCMV UL16, UL141, UL142, HIV Nef, HIV Vpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3, HTLV-1 p12, and cowpox virus protein CPXV203.

5. The chimeric protein construct according to claim 1, wherein the targeting domain comprises an antibody or a functional fragment thereof that specifically targets a target protein, and the functional fragment of the antibody is at least one selected from the group consisting of Fd, Fv, Fab, Fab', F(ab')2, Fv(scFv), single-chain antibody (scFv), nanobody, double-chain antibody, triple-chain antibody, and quadruple-chain antibody.

6. The chimeric protein construct according to claim 1, wherein the target protein is a pathogenic protein, and the pathogenic protein may be 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.

7. The chimeric protein construct according to claim 6, wherein the target protein is an immune function-related protein selected from antigen-presenting molecules (e.g., MHC class I molecules, MHC class II molecules, MICA / B molecules, etc.), antigen-recognition molecules (e.g., TCR, CD123, NKG2D, etc.), immune checkpoint molecules (e.g., PD-1, PD-L1, CTLA4, TIM3, TIGIT, LAG3, A2AR, BTLA, IDO1, IDO2, TDO, KIR, NOX2, VISTA, SIGLEC7, PVR, etc.), and immune-stimulating / co-stimulating molecules (e.g., CD3, CD80 / 86, CD28, etc.).

8. The chimeric protein construct according to claim 1, wherein the targeting domain does not specifically bind to the TCR.

9. The chimeric protein construct according to claim 8, 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, and preferably MHC II is HLA II.

10. The chimeric protein construct according to claim 1, wherein the targeting domain specifically binds to the TCR.

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

12. A chimeric protein construct according to claim 1, which is linked to at least one co-expression moiety.

13. The chimeric protein construct according to claim 12, wherein the at least one co-expressed moiety is independently selected from intact viral ER commensal glycoproteins (e.g., HCMV US2, US3, US11, US10, adenovirus E3-Kl9, HCMV US6, HSV ICP47), chimeric antigen receptors (CARs), functional T cell receptors (TCRs), chemokine receptors, and NK cell activating receptors.

14. The chimeric protein construct according to claim 13, wherein the chemokine receptor is selected from CCR4, CCR5, CCR6, CCR7, CCR9, CCR2b, CXCR1, CXCR2, and CXCR4.

15. The chimeric protein construct according to claim 13, wherein the NK cell activating receptor comprises (a) an extracellular domain (ED) of the NK cell activating receptor, (b) a transmembrane domain (TMD) of the NK cell activating receptor, and (c) an intracellular domain (ICD) of the NK cell activating receptor; a hinge or linker may be included between the extracellular domain of the NK cell activating receptor, the transmembrane domain of the NK cell activating receptor, and / or the intracellular domain of the NK cell activating receptor.

16. The chimeric protein construct according to claim 13, wherein the NK cell activation receptor is selected from NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, native cytotoxic receptor, TRAIL, DNAM-1, CD16a, 2B4, NTB-A, CRACC, and NKp80.

17. The chimeric protein construct according to claim 13, wherein the NK cell activating receptor forms a complex with a CNK signaling module.

18. The chimeric protein construct according to claim 12, wherein at least one co-expression portion is linked to the chimeric protein construct by a cleavable linker.

19. A nucleic acid molecule encoding the chimeric protein construct described in claim 1.

20. A vector comprising a nucleic acid molecule according to claim 19, wherein the nucleic acid molecule is operably linked to at least one polynucleotide regulator to express a chimeric protein construct encoded by the nucleic acid molecule.

21. An engineered cell expressing the chimeric protein construct described in claim 1, or comprising the nucleic acid molecule described in claim 19, or comprising the vector described in claim 20.

22. The engineered cell according to claim 21, wherein the cell is an immune cell selected from T cells, natural killer (NK) cells, B cells, macrophages, monocytes, dendritic cells, neutrophils, or γδT cells.

23. (i) a chimeric protein construct according to claim 1 or a nucleic acid molecule according to claim 19 or a vector according to claim 20 or an engineered cell according to claim 21, and (ii) a pharmaceutical composition or kit comprising a pharmaceutically acceptable medium.

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