WO2023227018A1 - Protéine de fusion ciblant des protéines de récepteur de membrane cellulaire et son utilisation - Google Patents

Protéine de fusion ciblant des protéines de récepteur de membrane cellulaire et son utilisation Download PDF

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WO2023227018A1
WO2023227018A1 PCT/CN2023/095988 CN2023095988W WO2023227018A1 WO 2023227018 A1 WO2023227018 A1 WO 2023227018A1 CN 2023095988 W CN2023095988 W CN 2023095988W WO 2023227018 A1 WO2023227018 A1 WO 2023227018A1
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amino acid
acid sequence
seq
variant
protein
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PCT/CN2023/095988
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Chinese (zh)
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刘凌峰
钟文婷
刘立琳
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羿尊生物医药（浙江）有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present disclosure relates to the field of therapy, and more specifically, to a fusion protein targeting a cell membrane receptor protein and its application.
  • Endocytosis is a mechanism by which small molecules or substrates are transferred into cells through cell membrane engulfment during cell operation. Endocytosis is usually divided into phagocytosis and pinocytosis, where phagocytosis refers to the endocytosis of larger solid particles (0.5–10 ⁇ m in diameter), such as pathogenic microorganisms. Pinocytosis is the internalization of various lipids through small endocytic vesicles. The endocytosis of pathogens or ligands binds to the cell membrane surface and forms endosomes (Phagosomes) or endocytic vesicles (endocytic vesicles) through receptor-dependent or receptor-independent mechanisms.
  • Membrane receptors bind to extracellular molecules and are responsible for conducting extracellular signals into cells. Studies have shown that some cell membrane receptors bind to ligands to form complexes, most of which are internalized and transferred to the cytoplasm or nucleus for signal transmission or degradation through the clathrin-dependent endocytosis mechanism (Aguilar RC et al., 2005).
  • T cell (antigen) receptors TCR
  • TTKs tyrosine receptor kinases
  • GPCRs G-protein coupled receptors
  • T cell (antigen) receptor TCR
  • TCR T cell (antigen) receptor
  • ⁇ / ⁇ or ⁇ / ⁇ Each chain is divided into a constant region and a variable region, and each chain contains a highly variable region CDR1 (complementarity-determining regions, CDR, complementary Determinant 1), CDR2, CDR3.
  • CDR1 and CDR2 are relatively conserved and are responsible for recognizing MHC;
  • CDR3 is the region where TCR directly contacts the antigen peptide and plays a decisive role in the interaction between TCR and the antigen peptide-MHC complex.
  • CDR3 is the most variable region, to a large extent. determines the diversity of TCR.
  • TCR In resting T cells, TCR will be continuously endocytosed and re-expressed on the cell surface. However, when TCR and pMHC combine, an immune complex synapse (Immune Synapse) of pMHC/TCR/CD3 will be formed. TCR endocytosis will rapidly increase and downregulate expression on the cell surface, reduce recycling, accelerate TCR degradation, and activate The signaling pathway within T cells stimulates T cell expansion or killing effect. During TCR activation, both bound TCR and surrounding unbound TCR can be internalized through Clathrin-dependent or -independent endocytosis. The internalized bound TCR/CD3 complex will be degraded, but most of the surrounding TCR/CD3 complex will recirculate and migrate to the immune synapse complex on the cell membrane surface.
  • CD3 antibody stimulation can also lead to TCR endocytosis, which is also a Clathrin-mediated endocytosis mechanism (Monjas, A., Alcover, A., and Alarco′n, B. (2004). Engaged and bystander T cell receptors are down -modulated by different endocytotic pathways.J.Biol.Chem.279,55376–55384.).
  • VXT is a pro-trimerizing domain derived from the trimerizing domain of human type XV collagen.
  • Collagen molecules are the main components of the extracellular matrix and play an important role in cell adhesion, movement, migration and other activities.
  • the XVT structure is derived from the trimer domain of human type XV or type XVIII collagen. This domain contains four ⁇ -sheet structures and ⁇ -helices, which can form a triple helix structure.
  • the present disclosure provides a method to target and bind the TCR structure on the surface of T cells by designing and expressing a recombinant protein containing a TCR-targeting domain and a trimerization-promoting domain, thereby achieving TCR structure aggregation and forming an immune synapse, thereby Induces the endocytosis of TCR structures to achieve the functions of targeted T cell directional labeling, delivery of drugs and nucleic acids, or detection or treatment.
  • the present disclosure also provides a method to target and bind CD3 on the surface of T cells by designing and expressing a recombinant protein containing a CD3-targeting domain and a trimerization-promoting domain, thereby achieving CD3 aggregation and inducing the endocytosis of CD3.
  • Target T cell directional labeling deliver drugs and nucleic acids, or achieve detection or treatment functions.
  • CD5 is a scavenger receptor family member protein that is constantly expressed on the membrane surface of T cells and some B1a class B cells. By treating T cells and B cells with anti-CD5 mAb, CD5 can be rapidly endocytosed (Xianghuai Lu et al., 2002). LNP targeting CD5 can efficiently deliver CAR mRNA into T cells and transform T cells into FAP CAR-T cells in vivo, which can eliminate fibroblasts activated during heart failure and achieve the purpose of treating cardiac fibrosis (Rurik JG et al., CAR T cells produced in vivo to treat cardiac injury.Science.2022Jan 7;375(6576):91-96.).
  • the present disclosure also provides a method to target and bind CD5 on the surface of T cells by designing and expressing a recombinant protein containing a CD5-targeting domain and a trimerization-promoting domain, thereby achieving CD5 aggregation and inducing the endocytosis of CD5.
  • Target T cell directional labeling deliver drugs and nucleic acids, or achieve detection or treatment functions.
  • CD7 is a 40KD single-domain Ig superfamily molecule transmembrane glycoprotein that is densely expressed on the surface of human differentiated mature T cells and NK cells and their precursor cells (BF Haynes et al., 1979, H Rabinowich et al., 1994) .
  • CD7 acts as a costimulatory factor for T cells by binding to its ligand K12/SecTM, plays an important role in T cell activation (Lyman SD et al., 2000).
  • CD7 is highly expressed on all T-cell acute lymphoblastic leukemia cells. Because CD7 can be rapidly endocytosed after binding to its ligands and antibodies without being cleaved and lost from the cell surface. Therefore, CD7 serves as an important target for ADC drugs to treat T lymphocyte leukemia.
  • the present disclosure also provides a method to target and bind CD7 on the surface of T cells by designing and expressing a recombinant protein containing a CD7-targeting domain and a trimerization-promoting domain, thereby achieving CD7 aggregation and inducing the endocytosis of CD7.
  • Target T cell directional labeling deliver drugs and nucleic acids, or achieve detection or treatment functions.
  • CD20 antigen also known as human B lymphocyte-restricted differentiation antigen, Bp35
  • Bp35 human B lymphocyte-restricted differentiation antigen
  • This antigen is also expressed on more than 90% of B-cell non-Hodgkin's lymphomas (NHL) (Blood63(6):1424-1433(1984)), but is not expressed on hematopoietic stem cells, primary B cells, normal plasma cells or other Not found in normal tissues (Tedder et al. J. Immunol. 135(2):973-979(1985)).
  • CD20 is thought to regulate an early step in the activation of cell cycle initiation and differentiation (Tedder et al., supra) and may function as a calcium channel (Tedder et al. J. Cell. Biochem. 14D:195( 1990)).
  • the present disclosure also provides a method to target and bind CD20 on the surface of T cells by designing and expressing a recombinant protein containing a CD20-targeting domain and a trimerization-promoting domain, thereby achieving CD20 aggregation and inducing the endocytosis of CD20.
  • Target T cell directional labeling deliver drugs and nucleic acids, or achieve detection or treatment functions.
  • CD22 is a 135 kDa B-cell-restricted sialoglycoprotein expressed only on the surface of differentiated mature B cells (Dorken, B. et al., J. Immunol. 136:4470-4479 (1986)) .
  • the most dominant form of CD22 in humans is CD22 ⁇ , which contains seven immunoglobulin superfamily domains in the extracellular domain ( Figure 1) (Wilson, G.L. et al., J. Exp. Med. 173:137-146( 1991)).
  • One variant form, CD22 ⁇ lacks immunoglobulin superfamily domains 3 and 4 (Stamenkovic, I. and Seed, B., Nature 345:74-77 (1990)).
  • the present disclosure also provides a method to target and bind CD22 on the surface of T cells by designing and expressing a recombinant protein containing a CD22-targeting domain and a trimerization-promoting domain, thereby achieving CD22 aggregation and inducing the endocytosis of CD22.
  • Target T cell directional labeling deliver drugs and nucleic acids, or achieve detection or treatment functions.
  • Fc receptor is a protein found on the surface of antigen-presenting cells, including B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, and basophils. cells, human platelets and mast cells, which help protect the function of the immune system.
  • the Fc receptor derives its name from its binding specificity for a portion of the antibody known as the Fc (crystallizable fragment) region.
  • Fc receptors bind to antibodies attached to infected cells or invading pathogens.
  • Fc receptors can stimulate phagocytes or cytotoxic cells to destroy microorganisms or infected cells through antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity.
  • Some viruses can increase virus invasion of cells (Antibody-Dependent Enhancement, ADE) through the mechanism of antibody-dependent enhancement of infection (Anderson R.Manipulation of cell surface macromolecules by flaviviruses. Adv Virus Res. 2003; 59:229- 274.doi:10.1016/s0065-3527(03)59007-8).
  • the present disclosure also provides a method to target and bind Fc receptors on the surface of T cells by designing and expressing a recombinant protein containing an Fc receptor-targeting domain and a trimerization-promoting domain, thereby achieving Fc receptor aggregation and inducing
  • the endocytosis of Fc receptors can achieve the functions of targeted T cell directional labeling, delivery of drugs and nucleic acids, or detection or treatment.
  • Figure 1 shows the basic structure of the fusion protein and its possible protein structural forms.
  • Figure 1A is the basic structure diagram of the fusion protein, including the targeting domain (Targeting Domain), the linker (Linker), and the trimer structure. Domain (Trimer domain);
  • Figure 1B is the basic structural diagram of the TCR-targeting fusion protein (BMA-STII-VXT), including the TCR-targeting single-chain antibody fragment (ScFv), Strep tag II tag, glyserine hinge region ( Glycine-Serine linker), collagen trimer domain (VXT);
  • Figure 1C is the basic structure diagram of the CD3-targeting fusion protein (BMC-STII-VXT), including the CD3-targeting single-chain antibody fragment (ScFv) , Strep tag II tag, Glycine-Serine linker, collagen trimer domain (VXT);
  • Figure 1D is the basic structure diagram of the CD5-targeting fusion protein (BMD-STII-VX
  • FIGlagen trimer domain (VXT);
  • Figure 1G is the basic structural diagram of the CD20-targeting fusion protein (BMG-STII-VXT), including the CD20-targeting single-chain antibody fragment (ScFv), Strep tag II tag, Glycine-Serine linker, collagen trimer domain (VXT);
  • Figure 1H is the basic structure diagram of the CD22-targeting fusion protein (BMH-STII-VXT), including a single chain targeting CD22 Antibody fragment (ScFv), Strep tag II tag, Glycine-Serine linker, collagen trimer domain (VXT);
  • Figure 1I shows the fusion protein targeting CD64 (BMI-STII-VXT) Basic structural diagram, including single-chain antibody fragment (ScFv) targeting CD64, Strep tag II tag, Glycine-Serine linker, and collagen trimer domain (VXT);
  • Figure 1J shows the possible fusion protein The protein structural forms: monomer, dimer (targeting
  • FIG. 2 shows the flow cytometric detection results of the specific binding of TCR-targeting fusion protein (BMA-STII-VXT) to T cells after Biotin modification.
  • BMA-STII-VXT TCR-targeting fusion protein
  • Figure 3 shows the flow cytometric detection results of T cells simultaneously labeled with TCR-targeting fusion protein (BMA-STII-VXT) and TCR-ab antibody.
  • Figure 3A shows that 95.5% of T cells show high expression of TCR- ⁇ .
  • Figure 3B shows that BMA-STII-VXT can completely shield TCR- ⁇ antibodies and compete for binding to T cells;
  • Figure 3C shows that BMA-STII-VXT can competitively bind to T cells, making TCR- ⁇ antibodies unable to bind to T cells;
  • Figure 3D shows BMA-VXT bound to TCR was internalized into T cells, and part of the TCR returned to the surface of T cells after 6 hours.
  • Figure 4 shows a schematic structural diagram of the connection between a TCR-targeting fusion protein and an LNP encapsulating nucleic acid inside.
  • Figure 5 shows a schematic structural diagram of the connection between a TCR-targeting fusion protein and an LNP containing a transposon internally.
  • Figure 6 shows a schematic structural diagram of the connection between a TCR-targeting fusion protein and an LNP entrapping a chemotherapeutic drug internally.
  • antibody refers to an immunoglobulin molecule that specifically binds to an antigen.
  • Antibodies can be intact immunoglobulins derived from natural or recombinant sources and can be immunoreactive portions of intact immunoglobulins.
  • Antibodies are usually tetramers of immunoglobulin molecules.
  • Antibodies in the present disclosure may exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Nanobodies, Fv, Fab, and F(ab), as well as single-chain, diabodies, tri-, and tetrabodies. and humanized antibodies.
  • an "antibody fragment” or “antigen-binding fragment” of an antibody refers to any portion of a full-length antibody that is less than full length but contains at least a portion of the variable region (e.g., one or more CDR and/or one or more antibody binding sites), and thus retains binding specificity and at least part of the specific binding ability of the full-length antibody.
  • an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds the same antigen as the antibody from which the antibody fragment is derived.
  • Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives.
  • Antibodies include antibody fragments.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , single chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, For example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov).
  • the fragments may include multiple chains linked together, for example by disulfide bonds and/or by peptide linkers.
  • Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids.
  • Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by displacing the corresponding region), results in an antibody that immunospecifically binds (i.e., exhibits a Ka of at least or at least about 10 7 -10 8 M ⁇ 1 ) for the antigen .
  • a "functional fragment” or “analog of an anti-CCR8 antibody” is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction.
  • functional fragments generally have the same meaning as "antibody fragments” and, with respect to antibodies, may refer to fragments that prevent or substantially reduce the ability of the receptor to bind a ligand or initiate signal transduction, e.g., Fv, Fab , F(ab') 2 and so on.
  • the "Fv” fragment consists of a dimer ( VH - VL dimer) formed by a non-covalent combination of the variable domain of a heavy chain and the variable domain of a light chain. In this configuration, the three CDRs of each variable domain interact to determine the target binding site on the surface of the VH - VL dimer, as is the case with intact antibodies. The six CDRs collectively confer the target binding specificity of the intact antibody.
  • variable domain or half of an Fv including only 3 target-specific CDRs
  • phrase "substantially identical" is understood to mean exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, Antibody chains with 97%, 98%, 99% or more sequence identity.
  • nucleic acid sequence the term is understood to mean exhibiting at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 %, 99% or higher sequence identity.
  • sequence identity or “identity” has art-recognized meanings, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule. Although there are many methods of measuring identity between two polynucleotides or polypeptides, the term “identity” is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988) ).
  • membrane receptor protein refers to a protein with specific functions on the cell membrane or within the cell, which can receive external signals and convert them into a series of biochemical reactions within the cell, causing changes in cell structure or function. There are monomer receptors and complex receptors.
  • vector is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell.
  • Many vectors are known in the art, including, but not limited to, linear polynucleotides, ionic or amphoteric related polynucleotides, plasmids, and viruses.
  • vector includes autonomously replicating plasmids or viruses.
  • the term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc.
  • viral vectors include, but are not limited to, lentivirus, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • non-viral vectors include, but are not limited to, CRISPR vector systems, Sleeping Beauty transposon systems, etc.
  • Activated refers to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector functions.
  • the term "activated T cells” refers in particular to T cells undergoing cell division.
  • the present disclosure provides a fusion protein comprising a module that binds to an antigen that enables clathrin-dependent endocytosis (CDE) and a module that promotes trimer formation.
  • CDE clathrin-dependent endocytosis
  • the cell membrane receptor protein is selected from the group consisting of T cell membrane receptor antigens, B cell membrane receptor antigens, and monocyte and other anti- One or more of the membrane receptor antigens of Antigen present cells.
  • the T cell membrane receptor antigen is selected from one or more of TCR, CD3, CD4, CD5, CD7, CCR5, and CXCR4.
  • the B cell membrane receptor antigen is selected from CD20 and CD22.
  • the monocyte and other antigen-presenting cell membrane receptor antigens are selected from Fc receptor antigens.
  • the Fc receptor antigen is selected from one or more of Fc ⁇ , Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIV, Fc ⁇ RI and Fc ⁇ / ⁇ R.
  • Fca is FcaRI, more preferably CD89.
  • FcyRII is CD64; preferably, FcyRII is FcyRIIB2 (CD32).
  • Fc ⁇ RI is CD89.
  • the aforementioned component that promotes trimer formation is selected from the group consisting of adiponectin or its collagen-like domain, T4 fibrin trimer domain, and the trimer structure of human collagen, such as: transmembrane N-terminal NC1 domain of collagens XIII, XV, XVII, and XVIII, NC2 domain of fibril-associated collagens IX, XII, XIV, XVI, agent protein A (SP-A), mannose-binding protein A (MBP-A), the catalytic subunit of Escherichia coli aspartate transcarbamylase (ATCase), oligomeric coiled-coil adhesins, and enveloped viruses Complementary heptad repeat region of class I fusion proteins.
  • SP-A agent protein A
  • MBP-A mannose-binding protein A
  • ATCase the catalytic subunit of Escherichia coli aspartate transcarbamylase
  • the component that promotes trimer formation is the amino-terminal NC1 domain of XV.
  • the component that promotes trimer formation includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 18, preferably 85%, 90%, 95%, An amino acid sequence having an identity of 96%, 97%, 98%, or 99% or more is more preferred, and an amino acid sequence having an identity of 98% or 99% or more is more preferred.
  • the component that promotes trimer formation comprises the amino acid sequence shown in SEQ ID NO. 18 or deletion, addition or substitution of 1, 2 or 3 residues on SEQ ID NO: 18. The resulting amino acid sequence.
  • the aforementioned component that binds to the antigen is selected from an antibody to the antigen or an antigen-binding fragment thereof or a ligand that binds to the membrane receptor protein.
  • the aforementioned TCR antigen antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO. 1 or any variant thereof, selected from the amino acid sequence SEQ ID NO. 2 or any variant thereof.
  • the heavy chain CDR2 of the variant is selected from the heavy chain CDR3 of the amino acid sequence SEQ ID NO. 3 or any variant thereof; and/or the light chain CDR1 is selected from the amino acid sequence SEQ ID NO. 4 or any variant thereof, selected from The light chain CDR2 of the amino acid sequence SEQ ID NO. 5 or any variant thereof is selected from the light chain CDR3 of the amino acid sequence SEQ ID NO. 6 or any variant thereof.
  • the antibody of the aforementioned TCR antigen or the antigen-binding fragment thereof comprises a CDR combination of heavy and light chains selected from the following:
  • the aforementioned TCR antibody fragment comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO. 7 or any variant thereof, and/or selected from the amino acid sequence SEQ ID NO. 8 or any variant thereof.
  • the light chain variable region of the body comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO. 7 or any variant thereof, and/or selected from the amino acid sequence SEQ ID NO. 8 or any variant thereof.
  • the antibody or antigen-binding fragment thereof of the aforementioned CD3 antigen comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO. 9 or any variant thereof, selected from the amino acid sequence SEQ ID NO. 10 or its The heavy chain CDR2 of any variant selected from the heavy chain CDR3 of the amino acid sequence SEQ ID NO. 11 or any variant thereof; and/or the light chain CDR1 selected from the amino acid sequence SEQ ID NO. 12 or any variant thereof, selected A light chain CDR2 selected from the amino acid sequence SEQ ID NO. 13 or any variant thereof.
  • the CD3 antibody or antigen-binding fragment thereof respectively comprises the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 9, 10, 11, and/or respectively comprises the amino acid sequence The light chain CDR1, CDR2 and CDR3 shown in SEQ ID NO:12, 13 and 14.
  • the CD3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO. 15 or any variant thereof, and/or selected from the amino acid sequence SEQ ID NO.
  • the light chain variable region of .16 or any variant thereof comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO. 15 or any variant thereof, and/or selected from the amino acid sequence SEQ ID NO.
  • the antibody or antigen-binding fragment thereof of the CD5 antigen comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO. 215 or any variant thereof, selected from the amino acid sequence SEQ ID NO. 216 or
  • the heavy chain CDR2 of any variant thereof is selected from the heavy chain CDR3 of the amino acid sequence SEQ ID NO. 217 or any variant thereof; and/or the light chain CDR1 is selected from the amino acid sequence SEQ ID NO. 218 or any variant thereof,
  • the antibody or antigen-binding fragment thereof of the CD5 antigen respectively comprises the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 215, 216, 217, and/or respectively comprises The amino acid sequence is the light chain CDR1, CDR2 and CDR3 shown in SEQ ID NO: 218, 219 and 220.
  • the antibody or antigen-binding fragment thereof of the CD5 antigen comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO. 221 or any variant thereof, and/or selected from the amino acid sequence SEQ The light chain variable region of ID NO. 222 or any variant thereof.
  • the antibody or antigen-binding fragment thereof of the CD7 antigen comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO. 223 or any variant thereof, selected from the amino acid sequence SEQ ID NO. 224 or
  • the heavy chain CDR2 of any variant thereof is selected from the heavy chain CDR3 of the amino acid sequence SEQ ID NO. 225 or any variant thereof; and/or the light chain CDR1 selected from the amino acid sequence SEQ ID NO. 226 or any variant thereof, Selected from The light chain CDR2 of the amino acid sequence SEQ ID NO. 227 or any variant thereof is selected from the light chain CDR3 of the amino acid sequence SEQ ID NO. 228 or any variant thereof.
  • the antibody or antigen-binding fragment thereof of the CD7 antigen respectively comprises the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 223, 224, 225, and/or respectively comprises The amino acid sequences are light chain CDR1, CDR2 and CDR3 shown in SEQ ID NO: 226, 227 and 228.
  • the antibody or antigen-binding fragment thereof of the CD7 antigen comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO. 229 or any variant thereof, and/or selected from the amino acid sequence SEQ The light chain variable region of ID NO. 230 or any variant thereof.
  • the antibody or antigen-binding fragment thereof of the CD4 antigen comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO. 231 or any variant thereof, selected from the amino acid sequence SEQ ID NO. 232 or
  • the heavy chain CDR2 of any variant thereof is selected from the heavy chain CDR3 of the amino acid sequence SEQ ID NO. 233 or any variant thereof; and/or the light chain CDR1 is selected from the amino acid sequence SEQ ID NO. 234 or any variant thereof,
  • the antibody or antigen-binding fragment thereof of the CD4 antigen respectively comprises the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 231, 232, 233, and/or respectively comprises The amino acid sequences are light chain CDR1, CDR2 and CDR3 shown in SEQ ID NO: 234, 235 and 236.
  • the antibody or antigen-binding fragment thereof of the CD4 antigen comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO. 237 or any variant thereof, and/or selected from the amino acid sequence SEQ The light chain variable region of ID NO. 238 or any variant thereof.
  • the antibody or antigen-binding fragment thereof of the CD20 antigen comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO. 239 or any variant thereof, selected from the amino acid sequence SEQ ID NO. 240 or
  • the heavy chain CDR2 of any variant thereof is selected from the heavy chain CDR3 of the amino acid sequence SEQ ID NO. 241 or any variant thereof; and/or the light chain CDR1 selected from the amino acid sequence SEQ ID NO. 242 or any variant thereof, A light chain CDR2 selected from the amino acid sequence SEQ ID NO. 243 or any variant thereof, a light chain CDR3 selected from the amino acid sequence SEQ ID NO. 244 or any variant thereof.
  • the antibody or antigen-binding fragment thereof of the CD20 antigen respectively comprises the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 239, 240, 241, and/or respectively comprises The amino acid sequences are light chain CDR1, CDR2 and CDR3 shown in SEQ ID NO: 242, 243 and 244.
  • the antibody or antigen-binding fragment thereof of the CD20 antigen comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO. 245 or any variant thereof, and/or selected from the amino acid sequence SEQ The light chain variable region of ID NO. 246 or any variant thereof.
  • the antibody or antigen-binding fragment thereof of the CD22 antigen comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO. 247 or any variant thereof, selected from the amino acid sequence SEQ ID NO. 248 or
  • the heavy chain CDR2 of any variant thereof is selected from the heavy chain CDR3 of the amino acid sequence SEQ ID NO. 249 or any variant thereof; and/or the light chain CDR1 is selected from the amino acid sequence SEQ ID NO. 250 or any variant thereof,
  • the antibody or antigen-binding fragment thereof of the CD22 antigen respectively comprises the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 247, 248, 249, and/or respectively comprises The amino acid sequences are light chain CDR1, CDR2 and CDR3 shown in SEQ ID NO: 250, 251 and 252.
  • the antibody or antigen-binding fragment thereof of the CD22 antigen comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO. 253 or any variant thereof, and/or selected from the amino acid sequence SEQ The light chain variable region of ID NO. 254 or any variant thereof.
  • the antibody or antigen-binding fragment thereof of the CD64 antigen comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO. 255 or any variant thereof, selected from the amino acid sequence SEQ ID NO. 256 or
  • the heavy chain CDR2 of any variant thereof is selected from the heavy chain CDR3 of the amino acid sequence SEQ ID NO. 257 or any variant thereof; and/or the light chain CDR1 selected from the amino acid sequence SEQ ID NO. 258 or any variant thereof, A light chain CDR2 selected from the amino acid sequence SEQ ID NO. 259 or any variant thereof, a light chain CDR3 selected from the amino acid sequence SEQ ID NO. 260 or any variant thereof.
  • the antibody or antigen-binding fragment thereof of the CD64 antigen respectively comprises the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 255, 256, 257, and/or respectively comprises
  • the amino acid sequences are the light chain CDR1, CDR2 and CDR3 shown in SEQ ID NO: 258, 259 and 260.
  • the antibody or antigen-binding fragment thereof of the CD64 antigen comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO. 261 or any variant thereof, and/or selected from the amino acid sequence SEQ The light chain variable region of ID NO. 262 or any variant thereof.
  • the component that binds to the antigen and the component that promotes trimer formation in the aforementioned fusion protein can be connected directly or through a linker L1.
  • the joint is a flexible joint.
  • the fusion protein also contains a purification tag; the purification tag is at the N-terminus, C-terminus of the fusion protein, or between the component that binds to the antigen and the component that promotes trimer formation. between.
  • the purification tag is between a component that binds the antigen and a component that promotes trimer formation.
  • the purification tag is selected from the group consisting of GST tag, His tag, Myc tag, E tag, Strep tag and HA tag.
  • the Strep tag is an STII tag, and its amino acid sequence is shown in SEQ ID NO. 17.
  • the flexible linker L1 is selected from GGGS (SEQ ID NO. 19), GGGGS (SEQ ID NO. 20), GGGGSGGGGS (SEQ ID NO. 21), SGGGGSGGGG (SEQ ID NO. 22) ,GGGGGSGGGGSSGGGGS(SEQ ID NO.23),GGGGGSGGGGSGGGGS(SEQ ID NO.24),GGGGSGGGGSGGGG(SEQ ID NO.25),GGGGSGGGGSGGGGSGGGGS(SEQ ID NO.26),GGGGSGGGGSGGGSGGGGS(SEQ ID NO.27),GSGSGS(SEQ ID NO.27) NO.28), GGSGSGSG(SEQ ID NO.29), GGSGSG(SEQ ID NO.30), GGSG(SEQ ID NO.31).
  • the linker L1 is selected from GGGS (SEQ ID NO. 19), GGGGSGGGGS (SEQ ID NO. 21), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO. 26), and GGGGSGGGGSGGGSGGGGS (SEQ ID NO. 27).
  • the fusion protein comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 36, preferably 85%, 90%, 95%, 96%, 97% , an amino acid sequence with 98% or more than 99% identity, more preferably an amino acid sequence with 98% or more than 99% identity; preferably, the amino acid sequence is the amino acid sequence shown in SEQ ID NO: 36 or in SEQ The amino acid sequence obtained by deleting, adding or substituting 1, 2 or 3 residues on ID NO:36.
  • the present disclosure provides nucleic acids encoding the aforementioned fusion proteins.
  • the present disclosure provides vectors comprising the aforementioned nucleic acids.
  • the present disclosure provides cells comprising the aforementioned nucleic acids or vectors.
  • the present disclosure provides a composition comprising the aforementioned fusion protein, the aforementioned nucleic acid, the aforementioned vector and/or the aforementioned cell.
  • the present disclosure provides a prodrug comprising any of the following:
  • the fusion protein and the LNP are connected through a linker L2; more preferably, the linker L2 is a polypeptide with a cysteine residue added to the C-terminus of the flexible polypeptide; the The flexible polypeptide is selected from the group consisting of GGGS (SEQ ID NO. 19), GGGGS (SEQ ID NO. 20), GGGGSGGGGS (SEQ ID NO. 21), SGGGGSGGGG (SEQ ID NO. 22), GGGGGSGGGGSSGGGGS (SEQ ID NO.
  • the linker L2 is selected from GGGSC (SEQ ID NO. 32), GGGGSGGGGSC (SEQ ID NO. 33), GGGGSGGGGSGGGGSGGGGSC (SEQ ID NO. 34) and GGGGSGGGGSGGGSGGGGSC (SEQ ID NO. 35).
  • the LNP contains at least one biologically active nucleic acid, protein and small molecule compound; preferably, the biologically active nucleic acid is selected from one or more of DNA and RNA; preferably, the small molecule compound is a chemotherapeutic drug; preferably, the chemotherapeutic drug is a cytotoxin.
  • the biologically active nucleic acid is selected from the group consisting of nucleic acids encoding chimeric antigen receptors (CARs), nucleic acids encoding CNK complexes, nucleic acids encoding pro-apoptotic proteins and nucleic acids encoding chimeric protein constructs.
  • CARs chimeric antigen receptors
  • CNK complexes nucleic acids encoding CNK complexes
  • pro-apoptotic proteins nucleic acids encoding pro-apoptotic proteins
  • nucleic acids encoding chimeric protein constructs One or more nucleic acids.
  • the biologically active nucleic acid is a nucleic acid encoding a transposon
  • the nucleic acid includes a nucleic acid of a target gene and a nucleic acid encoding a transposase.
  • the gene of interest is selected from one or more of the aforementioned chimeric protein receptors (CAR), chimeric natural killer cell receptor (CNK) complexes or chimeric protein constructs,
  • CAR chimeric protein receptors
  • CNK chimeric natural killer cell receptor
  • Figure 5 See Bonini C, Brenner MK, Heslop HE, Morgan RA. Genetic modification of T cells. Biol Blood Marrow Transplant. 2011; 17(1Suppl): S15-S20.doi:10.1016/ j.bbmt.2010.09.019).
  • the aforementioned chimeric antigen receptor includes an extracellular domain, a transmembrane domain, a costimulatory signaling region and an intracellular signaling domain, and the antigen bound by the extracellular domain Selected from: mesothelin (MSLN), B7-H3 (CD276), chondroitin sulfate proteoglycan 4 (CSPG4), Muc 16, Claudin 18.2, Claudin 8, NY-ESO-1, CD19, CD22, CD23, myeloid hyperplasia leukemia proteins MPL, CD30, CD32, CD20, CD70, CD99, CD123, CD138, CD179b, CD200R, CD324, Fc receptor-like 5FcRH5, CD171, CS-1 (signaling lymphocyte activation molecule family 7SLAMF7); the trans The membrane domain is the transmembrane region of CD8 or CD28; the costimulatory molecule is selected from MHC class I molecules, TNF receptor proteins,
  • SLAM protein activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, 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); the intracellular signaling domain is selected from CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), Fc ⁇ RI, CD66d, DAP10 and DAP12.
  • the present disclosure provides a multi-functional complex comprising the following components:
  • NK activating receptor component which at least includes NK cell activating receptor or functional variant thereof, said NK cell activating receptor including: (a) NK cell activating receptor extracellular domain (ED) or its function Variants, (b) NK cell activating receptor transmembrane domain (TMD) or functional variants thereof, and (c) NK cell activating receptor intracellular domain (ICD) or functional variants thereof; optionally, The NK cell activating receptor extracellular domain or its functional variant, the NK cell activating receptor transmembrane domain or its functional variant and/or the NK cell activating receptor intracellular domain or its function package between variants Contains hinges or joints;
  • CNK signal switching component which at least includes (i) an NK cell signal converter (adaptor) or a functional variant thereof, said NK cell signal converter comprising: (a) NK cell signal converter extracellular domain (ED) or functional variants thereof, (b) NK cell signal transducer transmembrane domain (TMD) or functional variants thereof, and (c) NK cell signal transducer intracellular domain (ICD) or functional variants thereof
  • a NK cell signal converter extracellular domain (ED) or functional variants thereof NK cell signal transducer transmembrane domain (TMD) or functional variants thereof
  • ICD NK cell signal transducer intracellular domain
  • a hinge or a linker is included between the NK activating receptor component and the CNK signal transfer component.
  • the NK cell activating receptor in the NK activating receptor component is selected from the group consisting of NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, a natural cytotoxic receptor body, TRAIL, DNAM-1, CD16a, 2B4, NTB-A, CRACC and NKp80; preferably, the natural cytotoxic receptor is selected from NKp46, NKp44 and NKp30.
  • the NK cell activating receptor is an NK cell activating receptor of mammalian origin; preferably, the mammal is selected from the group consisting of human, primate, mouse, horse, cow, sheep, Goats, cats, pigs, dogs, llamas, alpacas, elephants, squirrels, guinea pigs.
  • the NK cell activating receptor is a recombinant NK cell activating receptor comprising NK cell activating receptor domains from different sources.
  • the NK cell activating receptor is a human NK cell activating receptor; preferably, the NK cell activating receptor is a recombinant comprising different human NK cell activating receptor domains. NK cell activating receptor.
  • the NK cell activating receptor is a murine NK cell activating receptor; preferably, the NK cell activating receptor is a recombinant comprising different murine NK cell activating receptor domains. NK cell activating receptor.
  • the NK cell activating receptor is a recombinant NK cell activating receptor comprising human and murine NK cell activating receptor domains.
  • the extracellular domain of the NK cell activating receptor is the extracellular domain of a human or murine NK cell activating receptor.
  • the transmembrane domain of the NK cell activating receptor is that of a human or murine NK cell activating receptor.
  • the intracellular domain of the NK cell activating receptor is the intracellular domain of a human or murine NK cell activating receptor.
  • the functional variant of the NK cell activating receptor is selected from a mutant of the NK cell activating receptor, a wild-type fusion protein, or a fusion protein of a wild-type and a mutant type.
  • 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. 37, preferably 85%, 90%, 95%, 96%, 97 %, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the extracellular domain of human NKG2D is shown in SEQ ID NO. 37.
  • the full-length sequence of human NKG2D includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 38, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human NKG2D is shown in SEQ ID NO. 38.
  • 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. 39, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the extracellular domain of mouse NKG2D is shown in SEQ ID NO. 39.
  • 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: 40, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the full-length amino acid sequence of mouse NKG2D is shown in SEQ ID NO. 40.
  • 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: 41, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of human and mouse recombinant NKG2D is as shown in SEQ ID NO.41 Show.
  • the full-length sequence of human NKG2C includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 42, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human NKG2C is shown in SEQ ID NO. 42.
  • 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: 43, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human NKG2E is shown in SEQ ID NO. 43.
  • 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: 44, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human NKG2F is shown in SEQ ID NO. 44.
  • the full-length sequence of human CD94 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 45, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of human CD94 is shown in SEQ ID NO. 45.
  • 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: 46, preferably 85%, 90%, 95%, 96%, Amino acid sequences with more than 97%, 98%, or 99% identity, more preferably An amino acid sequence with an identity of 98% or more than 99%; the amino acid sequence of the full-length sequence of human KIR2DL4 is shown in SEQ ID NO. 46.
  • the full-length sequence of human KIR2DS1 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 47, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human KIR2DS1 is shown in SEQ ID NO. 47.
  • 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: 48, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human KIR2DS2 is shown in SEQ ID NO. 48.
  • the full-length sequence of human KIR2DS4 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 49, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human KIR2DS4 is shown in SEQ ID NO. 49.
  • the full-length sequence of human KIR3DS1 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 50, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human KIR3DS1 is shown in SEQ ID NO. 50.
  • the full-length sequence of human NKp46 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 51, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human NKp46 is shown in SEQ ID NO. 51.
  • the full-length sequence of human NKp44 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 52, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human NKp44 is shown in SEQ ID NO. 52.
  • the full-length sequence of human NKp30 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 53, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human NKp30 is shown in SEQ ID NO. 53.
  • the full-length sequence of human DNAM1 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 54, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human DNAM1 is shown in SEQ ID NO. 54.
  • 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: 55, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human TRAIL is shown in SEQ ID NO. 55.
  • 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: 56, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human CD16a is shown in SEQ ID NO. 56.
  • the full-length sequence of human 2B4 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 57, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human 2B4 is shown in SEQ ID NO. 57.
  • the full-length sequence of human NTB-A includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 58, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of human NTB-A is as shown in SEQ ID NO.58 Show.
  • 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: 59, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human CRACC is shown in SEQ ID NO. 59.
  • the full-length sequence of human NKp80 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 60, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human NKp80 is shown in SEQ ID NO. 60.
  • the NK cell signal transducer in the CNK signal transduction component is DAP10 or DAP12.
  • the NK cell signal transducer is a mammalian-derived NK cell signal transducer; preferably, the mammal is selected from the group consisting of human, primate, mouse, horse, cow, sheep, Goats, cats, pigs, dogs, llamas, alpacas, elephants, squirrels, guinea pigs.
  • the NK cell signal transducer is a recombinant NK cell signal transducer comprising NK cell signal transducer domains from different sources.
  • the NK cell signal transducer is a human NK cell signal transducer; preferably, the NK cell signal transducer is a recombinant comprising different human NK cell signal transducer domains. NK cell signal transducer.
  • the NK cell signal transducer is a murine NK cell signal transducer; preferably, the NK cell signal transducer is a recombinant comprising different murine NK cell signal transducer domains. NK cell signal transducer.
  • the NK cell signal transducer is a recombinant NK cell signal transducer comprising human and murine NK cell signal transducer domains.
  • the extracellular domain of the NK cell signal transducer is an extracellular structure of a human or murine NK cell signal transducer. area.
  • the transmembrane domain of the NK cell signal transducer is a transmembrane domain of a human or murine NK cell signal transducer.
  • the intracellular domain of the NK cell signal transducer is an intracellular domain of a human or murine NK cell signal transducer
  • the CNK cell signal transducer functional variant is selected from a DAP10 mutant or a mutant of DAP12, or a fusion protein of DAP10 and DAP12, or a combination of wild-type DAP10 or DAP12 and mutant DAP10 or DAP12 fusion protein.
  • the CNK signaling component further comprises (ii) an immunoreceptor activation signaling domain (ITAM) and/or (iii) a T cell costimulatory signaling domain.
  • ITAM immunoreceptor activation signaling domain
  • the NK cell signal transducer or functional variant thereof, the immunoreceptor activation signaling domain (ITAM) and/or the T cell costimulatory signaling domain comprise Hinge or linker;
  • the NK cell signal transducer or functional variant thereof is fused to the immune receptor activation signaling domain (ITAM) domain.
  • the immune receptor activation signaling domain is from an intracellular activation signaling domain of an immune receptor; preferably, the immune receptor is selected from TCR ⁇ , CD2, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, FcR ⁇ , CD66d, Fc ⁇ RI, Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIII, Dectin-1, CLEC-1, CD72, CD79A, CD79B;
  • the immune receptor activation signaling domain (ITAM ) is fused to an NK cell signal transducer or a functional variant thereof; preferably, the immune receptor is CD3 ⁇ .
  • the T cell costimulatory signaling domain is derived from the intracellular signaling domain of a costimulatory molecule; preferably, the costimulatory molecule is selected from the group consisting of MHC class I molecules, TNF receptor proteins, immune Globulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD16, CD27, CD28, CD30, CD40, CD38, CD35, CD79A, CD79B, CDS, ICAM-1, LFA-1, (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD
  • the full-length sequence of human DAP10 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 61, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human DAP10 is shown in SEQ ID NO. 61.
  • the full-length sequence of human DAP10 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 62, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human DAP10 is shown in SEQ ID NO. 62.
  • 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. 63, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the transmembrane domain of human DAP10 is shown in SEQ ID NO. 63.
  • the full-length sequence of human DAP12 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 64, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human DAP12 is shown in SEQ ID NO. 64.
  • 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. 65, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the transmembrane domain of human DAP12 is shown in SEQ ID NO. 65.
  • 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. 66, preferably 85%, 90%, Amino acid sequences with 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequences of the transmembrane domains of human DAP10 and human DAP12 are as follows SEQ ID NO.66 is shown.
  • 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. 67, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP10-DAP12 fusion protein sequence is shown in SEQ ID NO. 67.
  • 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. 68, preferably 85%, 90%, 95%, Amino acid sequences with more than 96%, 97%, 98%, and 99% identity sequence, more preferably an amino acid sequence with an identity of 98% or more than 99%; the amino acid sequence of the human CD3zeta intracellular signaling domain sequence is shown in SEQ ID NO. 68.
  • 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. 69, preferably 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP10-CD3zeta sequence is shown in SEQ ID NO. 69.
  • 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. 70, preferably 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP12-CD3zeta sequence is shown in SEQ ID NO. 70.
  • 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. 71, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP10-DAP12-CD3zeta sequence is shown in SEQ ID NO. 71.
  • 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. 72, preferably 85%, 90%, 95%, Amino acid sequences with 96%, 97%, 98%, or 99% or more identity, more preferably amino acid sequences with 98% or 99% or more identity; the amino acid sequence of the human 41BB intracellular signaling domain sequence is such as SEQ ID NO. 72 shown.
  • 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. 73, preferably 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP10-41BB sequence is shown in SEQ ID NO. 73.
  • 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. 74, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP10-41BB-CD3zeta sequence is shown in SEQ ID NO. 74.
  • 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. 75, preferably 85%, 90%, 95%, Amino acid sequences with 96%, 97%, 98%, or 99% or more identity, more preferably amino acid sequences with 98% or 99% or more identity; the amino acid sequence of the human CD28 intracellular signaling domain sequence is such as SEQ ID NO. 75 shown.
  • 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. 76, preferably 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP10-CD28 sequence is shown in SEQ ID NO. 76.
  • 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. 77, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP10-CD28-CD3zeta sequence is shown in SEQ ID NO. 77.
  • 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. 78, preferably 85%, 90%, 95%, 96%, 97 %, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP12-41BB sequence is shown in SEQ ID NO. 78.
  • 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. 79, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP12-41BB-CD3zeta sequence is shown in SEQ ID NO. 79.
  • 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. 80, preferably 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP12-CD28 sequence is shown in SEQ ID NO. 80.
  • 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. 81, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP12-CD28-CD3zeta sequence is shown in SEQ ID NO. 81.
  • the TCR-targeted binding protein molecule domain or functional variant thereof is derived from a TCR antibody or functional fragment thereof or a combination thereof.
  • the antibody is selected from TCR ⁇ antibody, TCR ⁇ antibody, TCR ⁇ antibody, TCR ⁇ antibody, TCR ⁇ antibody, TCR ⁇ antibody, TCR V ⁇ 2 antibody, TCR C ⁇ 1 antibody;
  • the functional fragment of the antibody is selected from Fd, Fv , Fab, Fab', F(ab')2, Fv (scFv), single-chain antibody (scFv) or nanobody (nanobody), diabody, three-chain antibody and four-chain antibody; preferably, the TCR antibody It is a TCR single chain antibody.
  • the ERAD degradation domain in the UT component is from HCMV glycoprotein US2, US3, US11 or US10, adenovirus E3-19K or HHV-7US21.
  • the full-length sequence of the HCMV glycoprotein US2 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 82, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US2 is such as SEQ ID Shown in NO.82.
  • 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. 83, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the HLA binding domain of the HCMV glycoprotein US2 is as follows SEQ ID NO.83 is shown.
  • 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. 84, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US2 is as follows SEQ ID NO.84 is shown.
  • the full-length sequence of the HCMV glycoprotein US3 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 85, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US3 is such as SEQ ID Shown in NO.85.
  • 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. 86, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the HLA binding domain of the HCMV glycoprotein US3 is as follows SEQ ID NO.86 is shown.
  • 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. 87, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US3 is as follows SEQ ID NO.87 is shown.
  • the full-length sequence of the HCMV glycoprotein US11 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 88, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US11 is such as SEQ ID Shown in NO.88.
  • the MHC binding domain of the HCMV glycoprotein US11 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 89, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the MHC binding domain of the HCMV glycoprotein US11 is as follows Shown in SEQ ID NO.89.
  • 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. 90, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US11 is as follows SEQ ID NO.90 is shown.
  • the full-length sequence of the HCMV glycoprotein US10 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 91, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US10 is such as SEQ ID Shown in NO.91.
  • 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. 92, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the HLA binding domain of the HCMV glycoprotein US10 is as follows Shown as SEQ ID NO.92.
  • 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. 93, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US10 is as follows SEQ ID NO.93 is shown.
  • the full-length sequence of the adenovirus E3-19K includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 94, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of adenovirus E3-19K is as follows Shown as SEQ ID NO.94.
  • the MHC binding domain of the adenovirus E3-19K comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 95, preferably 85%, 90%, Amino acid sequences with more than 95%, 96%, 97%, 98%, and 99% identity sequence, more preferably an amino acid sequence with an identity of 98% or more than 99%; the amino acid sequence of the MHC binding domain of the adenovirus E3-19K is shown in SEQ ID NO. 95.
  • the ERAD degradation domain of the adenovirus E3-19K comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 96, preferably 85%, 90%, Amino acid sequences with 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or 99% or more identity; amino acids of the ERAD degradation domain of the adenovirus E3-19K The sequence is shown as SEQ ID NO.96.
  • the full-length sequence of HHV-7US21 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 97, preferably 85%, 90%, 95%, An amino acid sequence with an identity of 96%, 97%, 98%, or 99% or more, and more preferably an amino acid sequence with an identity of 98% or more than 99%; the amino acid sequence of the full-length sequence of HHV-7US21 is such as SEQ ID NO. 97 shown.
  • the MHC binding domain of HHV-7US21 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 98, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the MHC binding domain of HHV-7US21 is such as SEQ ID Shown in NO.98.
  • the ERAD degradation domain of HHV-7US21 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 99, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the ERAD degradation domain of HHV-7US21 is such as SEQ ID Shown in NO.99.
  • the UT component further comprises (ii) a binding protein molecular domain or functional variant thereof that targets MHC I and/or MHC II.
  • the binding protein molecular domain or functional variant thereof targeting MHC I and/or MHC II is a binding protein molecular domain targeting HLA or a functional variant thereof.
  • the binding protein molecular domain or functional variant thereof targeting MHC I and/or MHC II is derived from a viral endoplasmic reticulum protein that inhibits the expression of MHC molecules or promotes their degradation; preferably, the The viral endoplasmic reticulum glycoprotein is selected from HCMV US6, HSV ICP47, CPXV012, HPV E6/E7, EBV BNFL2a or BHV UL49.5; preferably, the binding protein molecular domain targeting MHC I and/or MHC II or a functional variant thereof comprising a TAP binding domain.
  • the full-length sequence of HCMV US6 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 100, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of HCMV US6 is as shown in SEQ ID NO.100 Show.
  • the TAP binding domain of HHV-7US6 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 101, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the TAP binding domain of HHV-7US6 is such as SEQ ID Shown in NO.101.
  • the full-length sequence of HSV ICP47 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 102, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of HSV ICP47 is as shown in SEQ ID NO. 102 Show.
  • 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. 103, preferably 85%, 90%, 95%, An amino acid sequence with an identity of 96%, 97%, 98%, or 99% or more, and more preferably an amino acid sequence with an identity of 98% or more than 99%; the amino acid sequence of the TAP binding domain of the HSV ICP47 is such as SEQ ID NO. 103 shown.
  • 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. 104, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of CPXV012 is shown in SEQ ID NO. 104.
  • the TAP binding domain of CPXV012 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 105, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the TAP binding domain of CPXV012 is as shown in SEQ ID NO. 105 Show.
  • the full-length sequence of EBV BNFL2a includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 106, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of EBV BNFL2a is as shown in SEQ ID NO. 106 Show.
  • 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. 107, preferably 85%, 90%, 95%, An amino acid sequence with an identity of 96%, 97%, 98%, or 99% or more, more preferably an amino acid sequence with an identity of 98% or more than 99%; the amino acid sequence of the TAP binding domain of EBV BNFL2a is such as SEQ Shown as ID NO.107.
  • the full-length sequence of BHV UL49.5 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 108, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of BHV UL49.5 is such as SEQ ID Shown in NO.108.
  • the TAP binding domain of BHV UL49.5 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 109, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the TAP binding domain of the BHV UL49.5 is as follows Shown in SEQ ID NO.113.
  • the binding protein molecule domain or functional variant thereof targeting MHC I and/or MHC II is derived from a viral glycoprotein that degrades MHC and/or MHC II molecules; preferably, a viral glycoprotein Selected from HCMV glycoprotein US2, US3, US11 or US10, adenovirus E3-19K or HHV-7US21.
  • the full-length sequence of US2 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 110, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of US2 is shown in SEQ ID NO. 110.
  • the HLA binding domain of US2 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 111, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the HLA binding domain of US2 is as shown in SEQ ID NO. 111 Show.
  • the ERAD degradation domain of US2 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 112, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the ERAD degradation domain of US2 is as shown in SEQ ID NO. 112 Show.
  • the full-length sequence of US3 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 113, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of US3 is shown in SEQ ID NO. 113.
  • the HLA binding domain of US3 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 114, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the HLA binding domain of US3 is as shown in SEQ ID NO. 114 Show.
  • the ERAD degradation domain of US3 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 115, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the ERAD degradation domain of US3 is as shown in SEQ ID NO.115 Show.
  • the full-length sequence of US11 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 116, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of US11 is shown in SEQ ID NO. 116.
  • the HLA binding domain of US11 includes 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%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the HLA binding domain of US11 is as shown in SEQ ID NO.117 Show.
  • the ERAD degradation domain of US11 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 118, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the ERAD degradation domain of US11 is as shown in SEQ ID NO.118 Show.
  • the binding protein molecular domain or functional variant thereof targeting MHC I and/or MHC II further comprises targeted inhibition or degradation of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 or Viral protein of the NK target protein of ULBP6; preferably, the viral protein is selected from HCMV UL16, UL141, UL142 or adenovirus E3-19K.
  • the full-length sequence of HCMV UL16 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 119, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of HCMV UL16 is as shown in SEQ ID NO.119 Show.
  • the NK target protein binding domain of HCMV UL16 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 120, preferably 85%, 90%, An amino acid sequence with an identity of 95%, 96%, 97%, 98%, or 99% or more, and more preferably an amino acid sequence with an identity of 98% or more than 99%; the amino acid sequence of the NK target protein binding domain of the HCMV UL16 The amino acid sequence is shown in SEQ ID NO. 120.
  • 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. 121, preferably 85%, 90%, 95%, Amino acid sequences with 96%, 97%, 98%, or 99% or more identity, more preferably amino acid sequences with 98% or 99% or more identity; the amino acid sequence of the ERAD degradation domain of HCMV UL16 is such as SEQ ID NO. 121 shown.
  • the full-length sequence of HCMV UL141 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 122, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of HCMV UL141 is as shown in SEQ ID NO.122 Show.
  • the NK target protein binding domain of HCMV UL141 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 123, preferably 85%, 90%, Amino acid sequences with 95%, 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or more than 99% identity; the amino acids of the NK target protein binding domain of HCMV UL141 The sequence is shown as SEQ ID NO.123.
  • 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. 124, preferably 85%, 90%, 95%, Amino acid sequences with 96%, 97%, 98%, or 99% or more identity, more preferably amino acid sequences with 98% or more than 99% identity; the amino acid sequence of the ERAD degradation domain of HCMV UL141 is such as SEQ ID NO. 124 shown.
  • the full-length sequence of HCMV UL142 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 125, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of HCMV UL142 is as shown in SEQ ID NO.125 Show.
  • the MICA and ULBP3 binding domains of HCMV UL142 comprise an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 126, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequences of the MICA and ULBP3 binding domains of the HCMV UL142 are as follows Shown in SEQ ID NO.126.
  • the Golgi resident domain of HCMV UL142 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 127, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the Golgi resident domain of the HCMV UL142 is such as SEQ ID Shown in NO.127.
  • the binding protein molecular domain or functional variant thereof targeting MHC I and/or MHC II also includes a viral protein that transports MHC I molecules from the Golgi apparatus to lysosomes for degradation;
  • the viral protein is selected from HIV Nef, HIV Vpu, HHV-7U21, HHV-8KK3, HHV-8KK5, MHV-68MK3 and HTLV-1p12.
  • the HIV Nef comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 128, preferably 85%, 90%, 95%, 96%, 97% , an amino acid sequence with 98% or more than 99% identity, and more preferably an amino acid sequence with 98% or more than 99% identity; the amino acid sequence of HIV Nef is shown in SEQ ID NO. 128.
  • the HIV Vpu comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 129, preferably 85%, 90%, 95%, 96%, 97% , an amino acid sequence with 98% or more than 99% identity, and more preferably an amino acid sequence with 98% or more than 99% identity; the amino acid sequence of the HIV Vpu is shown in SEQ ID NO. 129.
  • the HHV-8KK3 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 130, preferably 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of HHV-8KK3 is shown in SEQ ID NO. 130.
  • the HHV-8KK5 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 131, preferably 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of HHV-8KK5 is shown in SEQ ID NO. 131.
  • the MHV-68MK3 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 132, preferably 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of MHV-68MK3 is shown in SEQ ID NO. 132.
  • the HTLV-1p12 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 133, preferably 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of HTLV-1p12 is shown in SEQ ID NO. 133.
  • the binding protein molecule domain or functional variant thereof targeting MHC I and/or MHC II also contains a protein that mediates the return of MHC-polypeptide molecules from the Golgi apparatus to the endoplasmic reticulum and promotes their degradation.
  • Viral protein Preferably, the viral protein includes an MHC binding structure and a KDEL receptor binding domain; Preferably, the viral protein is Cowpox Virus protein CPXV203.
  • the full-length sequence of the vaccinia virus protein CPXV203 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 134, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acids Sequence, more preferably an amino acid sequence with an identity of 98% or more than 99%; the amino acid sequence of the full-length sequence of the vaccinia virus protein CPXV203 is shown in SEQ ID NO. 134.
  • the MHC binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 135, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences;
  • the amino acid sequence of the MHC binding domain of the vaccinia virus protein CPXV203 is as follows Shown as SEQ ID NO.135.
  • the KDEL receptor binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 136, preferably 85%, 90% , 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the KDEL receptor binding domain of the vaccinia virus protein CPXV203 The amino acid sequence is shown in SEQ ID NO.136.
  • the full-length sequence of the vaccinia virus protein CPXV203 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 137, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of the vaccinia virus protein CPXV203 is such as SEQ ID Shown in NO.137.
  • the MHC binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 138, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences;
  • the amino acid sequence of the MHC binding domain of the vaccinia virus protein CPXV203 is as follows Shown in SEQ ID NO.138.
  • the KDEL receptor binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 139, preferably 85%, 90% , 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the KDEL receptor binding domain of the vaccinia virus protein CPXV203 The amino acid sequence is shown in SEQ ID NO.139.
  • the multifunctional complex further includes component (4) Chimeric adapter component and/or targeted killing tumor cell receptor component;
  • the (4) complex adapter includes: (i) a tumor-targeting extracellular recognition domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain; optionally, the Hinges or joints are included between tumor-targeting extracellular recognition domains, transmembrane domains, and/or intracellular signaling domains;
  • the tumor-targeting extracellular recognition domain of the composite adapter component is selected from the group consisting of a tumor antigen-specific binding domain, a tumor microenvironment target antigen-binding domain, and/or a chemotactic receptor targeting the tumor microenvironment. body.
  • the tumor-targeting extracellular recognition domain is selected from an antibody capable of targeting and recognizing a tumor-associated antigen or a functional fragment thereof, TCR, or a combination thereof; the functional fragment of the antibody is selected from Fd, Fv, Fab, Fab', F(ab')2, Fv(scFv), single chain antibody (scFv) or nanobody, diabody, tribody and quadrubody.
  • the transmembrane domain of the complex adapter component is selected from the group consisting of NK cell activating receptor transmembrane domain, DAP10 transmembrane domain, DAP12 transmembrane domain, CD8 transmembrane domain , CD28 transmembrane domain, CD4 transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, ICOS transmembrane domain, CTLA-4 transmembrane domain, PD-1 transmembrane domain, LAG -3 transmembrane domain, 2B4 transmembrane domain and BTLA transmembrane domain and combinations thereof; preferably, the NK cell activating receptor is selected from NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, natural cytotoxic receptors, TRAIL, DNAM-1, CD16
  • the intracellular signaling domain of the complex adapter component includes the intracellular signaling domain and/or the costimulatory signaling domain of an NK cell activating receptor.
  • the NK cell activating receptor is selected from NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, natural cytotoxic receptor, TRAIL, DNAM-1 , CD16a, 2B4, NTB-A, CRACC and NKp80.
  • the intracellular signaling domain also includes a costimulatory signaling domain; preferably, the costimulatory signaling domain is selected from T cell costimulatory signaling domains; including, but not Limited to derivatives derived from MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors body, 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, NKp
  • the targeted killing tumor cell receptor component includes (i) an extracellular recognition domain targeting tumor antigens; (ii) a transmembrane domain; and (iii) an intracellular costimulatory signaling domain; (iv) T Cell activation signaling domain (ITAM); optionally, the tumor antigen-targeting extracellular recognition domain, transmembrane domain, intracellular costimulatory signaling domain and/or T cell activation signaling domain (ITAM) contains hinges or joints;
  • the transmembrane domain targeting the tumor cell receptor component is selected from the group consisting of the CD8 transmembrane domain, the ⁇ and/or ⁇ chain transmembrane domain of the T cell receptor, the CD28 transmembrane domain, and the CD3 ⁇ transmembrane domain.
  • CD45 transmembrane domain CD4 transmembrane domain, CD5 transmembrane domain, CD8 transmembrane domain, CD9 transmembrane domain, CD16 transmembrane domain, CD22 transmembrane domain, CD33 transmembrane domain, CD37 Transmembrane domain, CD64 transmembrane domain, CD80 transmembrane domain, CD86 transmembrane domain, CD134 transmembrane domain, CD137 transmembrane domain, CD154 transmembrane domain, GITR transmembrane domain and combinations thereof;
  • the T cell activation signaling domains are derived from CD3 ⁇ , common FcR ⁇ (FCER1G), Fc ⁇ RIIa, FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD278 (“ICOS”), Fc ⁇ RI CD66d, DAP10 and DAP12 and other intracellular signaling domains.
  • the linker is a flexible linker; preferably, the flexible linker comprises the amino acid sequence shown (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 x and y are not both 0; more preferably, the linker includes the amino acid sequence shown in SEQ ID NO.140 or the amino acid sequence shown in SEQ ID NO.141 sequence.
  • the linker is a hinge; preferably, the hinge is an IgG1 hinge or an IgG4 hinge.
  • the IgG1 hinge comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 142, preferably 85%, 90%, 95%, 96%, 97% , an amino acid sequence with 98% or more than 99% identity, and more preferably an amino acid sequence with 98% or more than 99% identity; the amino acid sequence of the IgG1 hinge is shown in SEQ ID NO. 142.
  • the IgG4 hinge comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 143, preferably 85%, 90%, 95%, 96%, 97% , an amino acid sequence with 98% or more than 99% identity, and more preferably an amino acid sequence with 98% or more than 99% identity; the amino acid sequence of the IgG4 hinge is shown in SEQ ID NO. 143.
  • a cleavable peptide is included between the NK activating receptor component, CNK signal transduction component and/or UT component; for example, T2A peptide, GSG-T2A peptide, E2A peptide, GSG-E2A peptide , F2A peptide, GSG-F2A peptide, P2A peptide or GSG-P2A peptide.
  • the T2A includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 144, preferably 85%, 90%, 95%, 96%, 97%, An amino acid sequence with 98% or more than 99% identity, more preferably an amino acid sequence with 98% or more than 99% identity; the amino acid sequence of T2A is shown in SEQ ID NO. 144.
  • the amino acid sequence of the GSG-T2A peptide is as shown in SEQ ID NO. 145.
  • the P2A includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 146, preferably 85%, 90%, 95%, 96%, 97%, An amino acid sequence with 98% or more than 99% identity, more preferably an amino acid sequence with 98% or more than 99% identity; the amino acid sequence of P2A is shown in SEQ ID NO. 146.
  • the amino acid sequence of the GSG-P2A peptide is as shown in SEQ ID NO. 147.
  • the E2A includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 148, preferably 85%, 90%, 95%, 96%, 97%, An amino acid sequence with 98% or more than 99% identity, more preferably an amino acid sequence with 98% or more than 99% identity; the amino acid sequence of E2A is shown in SEQ ID NO. 148.
  • the amino acid sequence of the GSG-E2A peptide is as shown in SEQ ID NO. 149.
  • the F2A includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 150, preferably 85%, 90%, 95%, 96%, 97%, An amino acid sequence with 98% or more than 99% identity, more preferably an amino acid sequence with 98% or more than 99% identity; the amino acid sequence of F2A is shown in SEQ ID NO. 150.
  • the amino acid sequence of the GSG-F2A peptide is as shown in SEQ ID NO. 151.
  • the multifunctional complex includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 152, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the multifunctional complex includes as shown in SEQ ID NO. 152 TCR antibody single chain antibody.
  • the multifunctional complex includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 153, preferably 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the multifunctional complex is shown in SEQ ID NO. 153.
  • the present disclosure provides a nucleic acid molecule encoding the multifunctional complex.
  • the nucleic acid molecule is DNA or RNA.
  • the RNA is mRNA.
  • the nucleic acid molecule comprises a nucleotide sequence having 80% or more identity with the nucleotide sequence shown in SEQ ID NO. 154, preferably 85%, 90%, 95%, Nucleotide sequences with 96%, 97%, 98%, or 99% or more identity, more preferably 98% or more Amino acid sequences with more than 99% identity.
  • the present disclosure provides an expression vector containing the nucleic acid.
  • the vector is selected from plasmids, cosmids, viral vectors, RNA vectors or linear or circular DNA or RNA molecules.
  • the viral vector is selected from the group consisting of retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), elastic viruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., Mycovirus and Sendai), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses, including Adenoviruses, herpesviruses (eg, herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus) and poxviruses (eg, vaccinia, fowlpox, and canarypox), norovirus , togavirus, flavivirus, reovirus, papillomavirus,
  • retroviruses
  • the retrovirus is selected from the group consisting of avian leukocyte hyperplasia-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV collection, lentivirus, foamy viruses.
  • the lentiviral vector is selected from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or ovine demyelinating leukoencephalitis lentivirus.
  • the NK activating receptor component, CNK signal transduction component and/or UT component can be expressed in the same vector and the same promoter, or under different promoters, or in multiple vectors.
  • the vector is a lentiviral vector, and a cleavable peptide encoding gene is included between the genes encoding the NK activating receptor component, the CNK signal transduction component and/or the UT component; preferably, the The cleavable peptide is a 2A linker; the 2A linker is selected from T2A, P2A, E2A and F2A.
  • the vector further comprises a promoter; preferably, the promoter is an EF1 ⁇ promoter or a CMV promoter.
  • the present disclosure provides an immune cell comprising the nucleic acid or the expression vector.
  • the immune cells are selected from T cells, NKT cells, NK cells, B cells, monocytes, macrophages, etc.
  • the present disclosure provides a method of making immune cells, comprising introducing the nucleic acid or the expression vector into the cell using a method selected from: electroporation, sonoporation, gene gun (e.g., using Gene gun of Au-particles), lipofection, polymer transfection, nanoparticles or polyplexes.
  • the present disclosure provides a pharmaceutical composition comprising a multifunctional complex, the nucleic acid, the expression vector, the immune cell and/or the immune cell produced by the method, and Pharmaceutically acceptable carrier.
  • the present disclosure provides a multifunctional complex, the nucleic acid, the expression vector, the immune cell, the immune cell produced by the method and/or the pharmaceutical composition in preparation Use in medicines to treat diseases.
  • the present disclosure provides a method of treating a disease, comprising administering to a subject a multifunctional complex, the nucleic acid, the expression vector, the immune cell and/or the drug combination.
  • the diseases include various types of solid tumors and hematological tumors, viral infectious diseases, and autoimmune diseases.
  • the solid tumor is selected from the group consisting of 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.
  • nervous system tumors include diffuse glioma, diffuse astrocytoma and anaplastic astrocytoma, glioblastoma, oligodendroglioma, oligoastrocytoma tumors, childhood diffuse gliomas, other astrocytomas, ependymomas, neuronal and mixed neuronal-glial tumors, medulloblastoma, other embryonal tumors, schwannomas, meningiomas, Solitary fibrous tumor and hemangiopericytoma, etc.
  • head and neck tumors include malignant tumors of the nasal cavity and paranasal sinuses, nasopharyngeal cancer, oral cavity cancer, laryngeal cancer, salivary gland tumors, intracranial tumors, thyroid cancer, tongue cancer, etc.
  • thoracic tumors include lung cancer, esophageal cancer, cardiac cancer, breast cancer, mediastinal tumors, and the like.
  • digestive system tumors include gastric cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, small intestinal malignant tumors, and the like.
  • genitourinary tumors include kidney cancer, prostate cancer, bladder cancer, testicular malignancy, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, and the like.
  • soft tissue and skin tumors include malignant fibrous histiocytoma, rhabdomyosarcoma, synovial sarcoma, cutaneous malignant melanoma, and the like.
  • bone tumors include osteosarcoma, Ewing's sarcoma, and the like.
  • the colon cancer is a colon adenoma.
  • the breast cancer is triple negative breast cancer cells.
  • the liver cancer is hepatocellular carcinoma.
  • the disease is a hematological neoplasm selected from the group consisting of leukemia, lymphoma (HL), multiple myeloma (MM), myelodysplastic syndrome (MDS), and the like.
  • HL lymphoma
  • MM multiple myeloma
  • MDS myelodysplastic syndrome
  • the leukemia is B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute myeloid leukemia, etc.
  • viral infectious diseases include: respiratory viral diseases, gastrointestinal viral diseases, liver viral diseases, skin and mucosal viral diseases, eye viral diseases, central nervous system viral diseases, lymphatic Cellular viral diseases, insect-borne viral diseases, lentiviral infectious diseases, etc.
  • respiratory viral diseases include infections with rhinovirus, adenovirus, respiratory syncytial virus, parainfluenza virus, coronavirus, and the like; influenza; mumps, and the like.
  • gastrointestinal viral diseases include poliomyelitis; Cooksackie virus infection; ECHO virus infection; viral gastroenteritis: including rotavirus gastroenteritis, norovirus Gastroenteritis, adenovirus gastroenteritis, astrovirus gastroenteritis, coronavirus gastroenteritis and calicivirus gastroenteritis, etc.
  • the viral diseases of the liver include hepatitis A, hepatitis B, hepatitis C, hepatitis delta, hepatitis E, Epstein-Barr virus, and cytomegalovirus Viral hepatitis, etc.
  • viral diseases of the skin and mucosal membranes include measles, rubella, exanthema, varicella and shingles, smallpox, herpes simplex virus infection, rabies, foot and mouth disease, and the like.
  • ocular viral diseases include epidemic keratoconjunctivitis, follicular conjunctivitis, herpetic keratoconjunctivitis, and the like.
  • the central nervous system viral disease includes Japanese encephalitis, Western equine encephalitis, Eastern equine encephalitis, St. Louis encephalitis, Venezuelan equine encephalitis, Murray Valley encephalitis, California encephalitis inflammation, forest encephalitis and lymphocytic choriomeningitis.
  • lymphocytic viral diseases include infectious mononucleosis, cytomegalovirus infection, acquired immunodeficiency syndrome, and the like.
  • the insect-borne viral diseases include viral hemorrhagic fevers: including epidemic hemorrhagic fever, yellow fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, Argentine hemorrhagic fever, Venezuelan hemorrhagic fever, Lassa fever, Omsk hemorrhagic fever, Marburg disease and Ebola hemorrhagic fever, etc.; Dengue fever and dengue hemorrhagic fever; West Nile fever; Colorado tick-borne fever; sand fly fever, etc.;
  • lentiviral infectious diseases include subacute sclerosing panencephalitis, kuru disease, progressive multifocal leukoencephalopathy, subacute spongiform encephalopathy (corticostriatal spinal cord degeneration), and the like.
  • autoimmune diseases include organ-specific autoimmune diseases and systemic autoimmune diseases
  • organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, vulgaris Pemphigus, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, etc.
  • systemic autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune Hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, etc.
  • the present disclosure provides a method of stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of the multifunctional complex, the nucleic acid, the expression vector, the The immune cells, the immune cells produced by the method and/or the pharmaceutical composition.
  • the disclosure also provides cells, cell populations, and compositions (including pharmaceutical and therapeutic compositions) comprising the cells and populations, such as cells and populations produced by the provided methods, and methods, such as for administering the cells and methods of treating compositions to subjects, such as patients.
  • the pro-apoptotic protein is a Bcl-2 family protein, including but not limited to Bax, Bak, Bok, Bad, Bid, Bik, Bim, HrkBnip3, Nix/Bnip3L, Noxa and Puma.
  • the invention provides a chimeric protein construct for targeting a specific protein (i.e., target protein) to block its expression (e.g., surface expression) and pass through endoplasmic reticulum-associated degradation (ER -associated degradation (ERAD) mechanism accelerates ubiquitination-mediated degradation.
  • a specific protein i.e., target protein
  • ESD endoplasmic reticulum-associated degradation
  • Endoplasmic reticulum-associated protein degradation refers to a cellular pathway that targets misfolded proteins in the endoplasmic reticulum for ubiquitination and subsequent degradation by the proteasome.
  • the process of ERAD can be divided into three steps: (1) identification of misfolded or mutated proteins in the endoplasmic reticulum; (2) reverse transport of the identified misfolded or mutated proteins from the endoplasmic reticulum to the cytoplasm: terminal errors Folded proteins must be transported back from the endoplasmic reticulum to the cytoplasm, which contains the ubiquitin-proteasome system (UPS); (3) Use the proteasome to ubiquitin the recognized misfolded or mutated proteins Dependent degradation (see Annamaria et al., ER-associated degradation: Protein quality control and beyond. J. Cell Biol. Vol. 204 No. 6 869–879).
  • the present application provides a new chimeric strategy to fuse targeting protein binding domains (such as specific protein-targeting antibody fragments (ScFv)) to ERAD functional motifs (such as viral ER-resident proteins TMD and ED return signal domain), and demonstrated that this strategy can effectively inhibit the expression of specific proteins (for example, the expression of specific proteins on the cell surface).
  • targeting protein binding domains such as specific protein-targeting antibody fragments (ScFv)
  • ERAD functional motifs such as viral ER-resident proteins TMD and ED return signal domain
  • the TPD technology platform has the following advantages compared with existing protein degradation/removal technologies (such as PROTAC, CRISPR, etc.): 1.
  • the ER-TPD chimeric protein construct is passed through carriers (such as liposomes, nanoparticles, Lentivirus, adenovirus, oncolytic virus, etc.) are delivered to cells for stable expression and function.
  • carriers such as liposomes, nanoparticles, Lentivirus, adenovirus, oncolytic virus, etc.
  • ER-TPD technology is based on endoplasm
  • the ERAD principle of the reticulum directly intervenes in protein synthesis and achieves efficient degradation during the endoplasmic reticulum protein synthesis and assembly process.
  • the targeting domain can adopt various single-chain antibody structures or artificial affinity ligand structures, which greatly realizes the targeting of target proteins. Specific recognition and binding ability; while conventional PROTAC technology uses the chemical structure of small molecules, it is difficult to ensure the specificity of the targeting structure, so ER-TPD technology has the powerful advantage of targeting accuracy and specificity; 4.
  • ER-TPD technology can realize the multi-pathway mechanism of target protein degradation through the downstream degradation-directed ligand structure, including the UPS mechanism based on ubiquitin proteasome and the ALP mechanism of autophagy lysosome.
  • the design of the downstream degradation-directed ligand structure can Through genetic engineering methods, flexibly connect the intracellular degradation system to achieve efficient targeted degradation, while conventional PROTAC technology can only be degraded by targeting E3 ligase; 5.
  • TPD technology degrades target proteins at the protein level. Compared with technologies that block the expression of target proteins at the gene level (such as CRISPR technology and siRNA technology), there is no off-target error. Chromosomal instability and other toxic side effects caused by ER-TPD technology or gene editing process; 6.
  • the present disclosure provides a chimeric protein construct (chimeric protein construct) based on endoplasmic reticulum-based Targeted Protein Degradation (ER-TPD) technology, which includes endoplasmic reticulum ER-associated degradation (ERAD) mechanism protein binding domain and targeting protein binding domain.
  • ER-TPD Endoplasmic reticulum-based Targeted Protein Degradation
  • ESD endoplasmic reticulum ER-associated degradation
  • the chimeric protein construct further comprises a protein degradation pathway member (eg, a ubiquitin-proteasome system pathway member, an endosome-lysosome pathway member, an autophagy pathway member) binding domain.
  • a protein degradation pathway member eg, a ubiquitin-proteasome system pathway member, an endosome-lysosome pathway member, an autophagy pathway member
  • the protein degradation pathway member binding domain may be linked to the constituent elements of the chimeric protein construct in any suitable manner, for example, to the ERAD machinery protein binding domain.
  • the present disclosure also provides a library of compositions, wherein each composition comprises a viral protein or a nucleic acid molecule encoding the same, which comprises a protein capable of hijacking the ERAD machinery to block and accelerate proteasome-mediated degradation within the ER.
  • Functional motifs and affinity moieties that target specific proteins for degradation are used for therapeutic purposes to modify cell phenotype, improve cell function, or induce apoptosis or inhibit viral replication.
  • ERAD mechanism protein refers to a protein that participates in the ERAD mechanism or pathway.
  • ERAD machinery protein binding domain also known as “ERAD degradation domain” refers to a portion or domain capable of binding and/or utilizing an ERAD machinery protein, e.g., a viral endoplasmic reticulum (ER)-resident glycoprotein.
  • ER viral endoplasmic reticulum
  • transmembrane domain or a functional variant thereof
  • cytoplasmic domain or a functional variant thereof
  • the ERAD machinery protein binding domain comprises a transmembrane domain of a viral endoplasmic reticulum resident protein, or a functional variant thereof, and an endoplasmic reticulum resident domain, or a functional variant thereof.
  • the viral ER-resident glycoprotein can be any viral ER-resident protein that is capable of hijacking the ERAD machinery and promoting ubiquitination and proteasome-mediated degradation of the target protein.
  • the viral endoplasmic reticulum resident protein is adenovirus E3-19K.
  • Adenovirus E3-19K (also known as "E19") contains three functional modules: a luminal domain for interaction with MHC-I and MICA/B molecules, a transmembrane domain, and an endoplasmic reticulum-resident domain, in which Its endoplasmic reticulum-resident domain contains a dilysine motif in the cytoplasmic tail that returns the Golgi apparatus to the endoplasmic reticulum, so it can also be called the "ER return signal motif.”
  • ER return signal motif adenovirus E3-19K
  • transmembrane domains and ER return signaling motifs are required to ensure efficient ER localization, histocompatibility complex class I (MHC-I) and MHC-I-associated chains A and B (MICA/B molecules) Transport inhibition and proteasomal degradation.
  • adenovirus E3-19K can host MHC class I molecules (e.g., MHC-I and MICA/B molecules) in the secretory pathway and interfere with antigen presentation.
  • MHC class I molecules e.g., MHC-I and MICA/B molecules
  • the chimeric protein construct provided by the present disclosure utilizes the transmembrane domain and endoplasmic reticulum resident domain (ie, ERAD degradation domain) of provirus E3-19K to fuse with the target protein binding domain to achieve degradation of the target protein. Purpose.
  • the ERAD degradation domain of the adenovirus E3-19K comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 96, preferably 85%, 90%, An amino acid sequence having an identity of 95%, 96%, 97%, 98%, or 99% or more is more preferred, and an amino acid sequence having an identity of 98% or 99% or more is more preferred.
  • the ERAD degradation domain of adenovirus E3-19K has a dilysine motif in the cytoplasmic tail, which can return the Golgi apparatus to the endoplasmic reticulum.
  • the amino acid sequence of the ERAD degradation domain of the adenovirus E3-19K is shown in SEQ ID NO. 96.
  • the viral endoplasmic reticulum resident protein is not adenovirus E3-19K.
  • the viral endoplasmic reticulum resident protein is selected from at least one of the following: HCMV glycoprotein US2, US11, US3, US10, US6, HSV ICP47, CPXV12, BHV UL49.5, EBV BNFL2a, HCMV UL16, UL141, UL142, HIV Nef, HIV Vpu, HHV-7U21, HHV-8KK3, HHV-8KK5, MHV-68MK3, HTLV-1p12 and Cowpox Virus protein CPXV203.
  • the viral endoplasmic reticulum resident protein comprises HCMV glycoproteins US2 and US11.
  • the viral endoplasmic reticulum resident protein comprises E3-19K and at least one selected from the group consisting of: HCMV glycoprotein US2, US11, US3, US10, US6, HSV ICP47, CPXV12, BHV UL49.5 , EBV BNFL2a, HCMV UL16, UL141, UL142, HIV Nef, HIV Vpu, HHV-7U21, HHV-8KK3, HHV-8KK5, MHV-68MK3, HTLV-1p12 and Cowpox Virus protein CPXV203.
  • US2 and US11 bind to MHC class I heavy chains through their luminal domains and recruit host cell proteins, which extract polypeptides from the endoplasmic reticulum membrane by "pulling" the cytoplasmic tails of the heavy chains. After MHC class I molecules are translocated into the cytoplasm, they are ubiquitinated and degraded by the proteasome.
  • US2 leads to the degradation of two proteins of the class II pathway, DR- ⁇ and DM- ⁇ , and HFE, a nonclassical major histocompatibility complex (MHC) class I protein involved in iron regulation. .
  • MHC major histocompatibility complex
  • the luminal domain of US2 is responsible for binding MHC class I and class II molecules, and the transmembrane domain (TM) and cytoplasmic domain (CT) interact with cellular components of the ERAD machinery or pathway and contribute to translocation and promotion of MHC I Enzymatic hydrolysis of both class II and class II proteins (Chevalier M S et al., 2002, 2003).
  • the cytoplasmic tail of US2 is sufficient to interact with the signal peptide peptidase (SPP), a US2-dependent MHC I translocation complex (US2- dependent MHC I dislocation complex) (Loureiro J et al., 2006) and is necessary for US2-dependent MHC heavy chain translocation (see Joana et al., Signal peptide peptidase is required for dislocation from the endoplasmic reticulum. Nature volume 441, pages 894–897(2006)).
  • SPP signal peptide peptidase
  • US2-dependent MHC I dislocation complex US2-dependent MHC I dislocation complex
  • US2 interacts with the endoplasmic reticulum-resident RING-type E3 ligase TRC8 through its TM domain, which also contributes to the ubiquitination and proteasomal degradation of US2 tail-anchored MHC I and II molecules (Stagg H R et al. ,2009).
  • US11-induced degradation of MHC-I molecules requires Derlin-1 but not SPP.
  • the ER luminal domain of US11 interacts with the luminal domain of 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 B N et al., 2004; Cho S et al., 2013) and then induce their translocation to the cytosol for proteasomal processing degradation.
  • US11 activates unfolded proteins. Through Derlin-1, US11 associates with TMEM129 as an ERAD RING E3 ligase and recruits Ube2J2 to ubiquitinate MHC-I prior to US11-induced degradation.
  • 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. 84, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or above identity of the amino acid sequence, more preferably 98% or 99% or above identity of the amino acid sequence, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US2 is shown in SEQ ID NO. 84.
  • the ERAD degradation domain of US2 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 112, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or above identity of the amino acid sequence, more preferably 98% or 99% or more identity of the amino acid sequence, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of the ERAD degradation domain of US2 is shown in SEQ ID NO. 112.
  • 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. 90, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or above identity of the amino acid sequence, more preferably 98% or 99% or above identity of the amino acid sequence, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US11 is shown in SEQ ID NO. 90.
  • the ERAD degradation domain of US11 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 118, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or above identity of the amino acid sequence, more preferably 98% or 99% or more identity of the amino acid sequence, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of the ERAD degradation domain of US11 is shown in SEQ ID NO. 118.
  • the HCMV US3 glycoprotein physically binds to peptide-loaded MHC class I heterodimers, resulting in retention of class I complexes in the ER and inhibition of invariant chain interaction with class II DR- ⁇ in the ER. Binding of dimers leads to mislocalization of class II complexes and reduced peptide loading. Therefore, US3 is able to interfere with the intracellular transport and maturation of MHC class I molecules during the early stages of HCMV infection. US3 is an endoplasmic reticulum-resident membrane protein.
  • 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. 87, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or above identity of the amino acid sequence, more preferably 98% or 99% or above identity of the amino acid sequence, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US3 is shown in SEQ ID NO. 87.
  • the ERAD degradation domain of US3 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 115, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or above identity of the amino acid sequence, more preferably 98% or 99% or more identity of the amino acid sequence, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of the ERAD degradation domain of US3 is shown in SEQ ID NO. 115.
  • the HCMV US10 glycoprotein also interacts with components of MHC class I antigen presentation. US10 binds free class I heavy chains and delays their transport from the ER. However, US10 does not affect US2 or US11.
  • 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. 93, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or above identity of the amino acid sequence, more preferably 98% or 99% or above identity of the amino acid sequence, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US10 is shown in SEQ ID NO. 93.
  • HCMV L protein US6 affects antigen presentation through a completely different strategy.
  • US6 inhibits the translocation of cytosolic peptides through the TAP complex (TAP1/2).
  • TAP1/2 TAP complex
  • US6 binds to the ER luminal side of TAP1 and causes a conformational change, thereby preventing ATP binding.
  • Residues 89-108 in the ER-luminal domain of US6 contribute to US6 binding to TAP and are necessary and sufficient for this inhibition.
  • This inhibition of TAP activity affects not only the expression of classical MHC class I alleles but also the expression of non-classical alleles HLA-C and HLA-G in fetal cytotrophoblast cells.
  • HSV ICP47 As the HCMV US6 protein, HSV ICP47 is expressed early in the infection cycle and is dispensable for in vitro replication, the same strategy can also be applied to prevent class I molecule assembly. ICP47 blocks TAP-mediated peptide transport and binds tightly to the TAP1-TAP2 complex. One clue that ICP47 blocks the TAP mechanism is that it exhibits high species selectivity. Both HSV1 and HSV2I CP47 inhibit simian, monkey, pig, dog, and bovine TAP and have little effect on mouse, rat, guinea pig, or rabbit TAP. ICP47 has approximately 100-fold higher affinity for human TAP than for mouse TAP. ICP47 inhibitory peptide and TAP Binding, but does not affect ATP binding. ICP47 has an affinity for TAP that is 10-1000 times higher than most peptides, acts as a competitive inhibitor of peptide binding to TAP, and is thought to bind directly to the peptide binding site.
  • the TAP binding domain of HHV-7US6 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 101, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of the TAP binding domain of HHV-7US6 is shown in SEQ ID NO. 101.
  • 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. 103, preferably 85%, 90%, 95%, An amino acid sequence with an identity of 96%, 97%, 98%, or 99% or more, more preferably an amino acid sequence with an identity of 98% or 99% or more, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of the TAP binding domain of HSV ICP47 is shown in SEQ ID NO. 103.
  • the TAP binding domain of CPXV012 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 105, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or above identity of the amino acid sequence, more preferably 98% or 99% or more identity of the amino acid sequence, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of the TAP binding domain of CPXV012 is shown in SEQ ID NO. 105.
  • 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. 107, preferably 85%, 90%, 95%, An amino acid sequence with an identity of 96%, 97%, 98%, or 99% or more, more preferably an amino acid sequence with an identity of 98% or 99% or more, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of the TAP binding domain of EBV BNFL2a is shown in SEQ ID NO. 107.
  • the TAP binding domain of BHV UL49.5 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 109, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or above identity of the amino acid sequence, more preferably 98% or 99% or above identity of the amino acid sequence, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of the TAP binding domain of BHV UL49.5 is shown in SEQ ID NO. 109.
  • the ERAD degradation domain of HHV-7US21 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 99, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of the ERAD degradation domain of HHV-7US21 is shown in SEQ ID NO. 99.
  • 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. 121, preferably 85%, 90%, 95%, An amino acid sequence with an identity of 96%, 97%, 98%, or 99% or more, more preferably an amino acid sequence with an identity of 98% or 99% or more, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of the ERAD degradation domain of HCMV UL16 is shown in SEQ ID NO. 121.
  • 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. 124, preferably 85%, 90%, 95%, An amino acid sequence with an identity of 96%, 97%, 98%, or 99% or more, more preferably an amino acid sequence with an identity of 98% or 99% or more, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of the ERAD degradation domain of HCMV UL141 is shown in SEQ ID NO. 124.
  • the Golgi resident domain of HCMV UL142 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 127, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of the Golgi-resident domain of HCMV UL142 is set forth in SEQ ID NO. 127.
  • the HIV Nef comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 128, preferably 85%, 90%, 95%, 96%, 97% , an amino acid sequence with 98% or more than 99% identity, more preferably an amino acid sequence with 98% or more than 99% identity, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of HIV Nef is set forth in SEQ ID NO. 128.
  • the HIV Vpu comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 129, preferably 85%, 90%, 95%, 96%, 97% , an amino acid sequence with 98% or more than 99% identity, more preferably an amino acid sequence with 98% or more than 99% identity, and has the function of endoplasmic reticulum residence.
  • the HIV Vpu has an amino acid sequence as shown in SEQ ID NO. 129.
  • the HHV-8KK3 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 130, preferably 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of HHV-8KK3 is shown in SEQ ID NO. 130.
  • the HHV-8KK5 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 131, preferably 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of HHV-8KK5 is set forth in SEQ ID NO. 131.
  • the MHV-68MK3 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 132, preferably 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of MHV-68MK3 is shown in SEQ ID NO. 132.
  • the HTLV-1p12 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 133, preferably 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences, and has the function of endoplasmic reticulum residence.
  • the amino acid sequence of HTLV-1p12 is shown in SEQ ID NO. 133.
  • the KDEL receptor binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 136, preferably 85%, 90% , 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences, and have the function of endoplasmic reticulum residence.
  • the amino acid sequence of the KDEL receptor binding domain of the vaccinia virus protein CPXV203 is shown in SEQ ID NO. 136.
  • the KDEL receptor binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 139, preferably 85%, 90% , 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences, and have the function of endoplasmic reticulum residence.
  • the amino acid sequence of the KDEL receptor binding domain of the vaccinia virus protein CPXV203 is shown in SEQ ID NO. 139.
  • the target binding domain of the chimeric protein construct provided by the present disclosure can be any structure that recognizes and binds the target protein, such as antibodies, antibody fragments, functional motifs derived from natural proteins (e.g., natural ligands ), artificially synthesized polypeptides or proteins with affinity for the target protein, or various variant forms such as mutants, fusions, truncations, etc. of the above molecules.
  • the targeting domain can be a natural ligand of the target protein, an antibody that specifically recognizes the target protein, or an antigen-binding fragment thereof, or it can be an antigen that can be specifically recognized by an antibody.
  • the targeting domain comprises an antibody or functional fragment thereof that specifically targets a target protein (e.g., diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, di- Sulfide bond stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide bond stabilized diabody (ds diabody), single chain antibody molecule (scFv), scFv Dimer (bivalent bifunctional antibody), Fv (scFv), multispecific antibody (e.g., bispecific antibody), camelized single domain antibody (e.g., VHH), Nanobody, domain antibody, bivalent Domain antibodies, diabodies, tribodies and quadribodies.
  • the targeting protein domain can also include any binding ligand capable of binding to the target protein, including but not limited to, ligands, receptor binding ligands part of the viral protein, the part of the viral protein that binds to the target protein
  • the target protein can be any protein whose level or activity is desired to be modulated. For example, changing activity through the ERAD mechanism to block proteins in the ER and accelerate proteasome-mediated degradation, whose degradation can change the phenotype of the cell and improve cell function. , causing cell apoptosis and/or inhibiting virus replication, etc., thereby achieving therapeutic purposes (for example, improving efficacy, reducing side effects, etc.).
  • the target protein may be involved in cell cycle, apoptosis, signal transduction, cell differentiation, cell dedifferentiation, cell growth, production of cytokines or biological regulators thereof, production of cytokines or biological regulators, Proteins that regulate or function in protein production, pro-inflammatory signaling, or glucose regulatory pathways.
  • the target protein is a disease-associated protein (also referred to as a "pathogenic protein")
  • disease-associated protein also referred to as a "pathogenic protein”
  • pathogenic protein refers to any protein whose function or activity would result in The occurrence of disease, or proteins whose functions are important for the development of disease.
  • pathogenic proteins include, but are not limited to, oncogenic proteins, viral proteins, and autoimmune disease-causing proteins (for example, antibodies produced by plasma cells).
  • the target protein is selected from: immune function-related target proteins, neurological disease-related target proteins, infectious disease-related target proteins (such as viral infection-related target proteins), autoantigen-related Target proteins or tumor-related target proteins (or oncogenic proteins), metabolic disease-related target proteins.
  • the disease-causing protein is an oncogenic protein.
  • the oncogenic protein may be encoded by an oncogene, including but not limited to BCL2, c-MYC, Ras, HER2, BCR/ABL, ABL1/BCR, TGFB1, TLX1, P53, WNT1, WNT2, WT1, ⁇ v- ⁇ 3, 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
  • the oncogenic protein can be a Bcl-2 family member (such as: Bcl-2, Bcl-xL and Bcl-w), VEGF/VEGFR, PDGFR ⁇ , EGFR, EGFR mutants, IGF-1R, HDACs, HER2, MYC, KRAS, AFP, CEA, CA199, estrogen receptor (estrogen receptor ER- ⁇ ), androgen receptor (AR), tyrosine kinase (c-ABL, BCR-ABL, BTK, FAK, PTK6, Wee1, TRK transmembrane receptor), serine/threonine kinase receptor (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, PB
  • the target protein includes an immune function-related protein.
  • immune function-related proteins refers to functional proteins involved in the body's immune process, such as antigen presentation molecules (such as MHC class I molecules, MHC class II molecules, HLA, etc.), antigen recognition molecules (such as TCR, CD123 , NKG2D, etc.), immune checkpoint molecules (such as PD-1, PD-L1, CTLA4, TIM3, TIGIT, LAG3, A2AR, BTLA, IDO1, IDO2, TDO, KIR, NOX2, VISTA, SIGLEC7, PVR, etc.), immune Stimulatory/co-stimulatory molecules (e.g.
  • immune function-related target proteins include, for example, CD123, CD7, CD5, MHC class I molecules, MHC class II molecules, non-classical MHC molecules (such as HLA-G, HLA-E), MICA/B, ULBP1-6, IL6, IL6 receptor, IL1 receptor, RANKL, TGF- ⁇ 1, PD1, PD-L1, CTLA4, Tim3, LAG3, Siglec-15, TIGIT, CD47, IL4RA, CD94/NKG2A, CXCR1/2, CXCL8, CCR2/CCR5, CCR4, CXCR4, c-Rel, CCL2, CCL5, CCL20, CCL22, CSF-1, CCL2, CCL5 indoleamine-2,3-dioxygenase (IDO), or arginase 1(ARG1).
  • MHC class I molecules such as HLA-G, HLA-E
  • MICA/B such as HLA-G, HLA-E
  • ULBP1-6 ULBP1-6
  • the target protein includes a target protein associated with a neurological disease.
  • the term "neurological disease associated” as used herein refers to proteins involved in neurological diseases, particularly in neurodegenerative diseases.
  • the target protein associated with neurological disease includes, for example, Tau, amyloid- ⁇ (A ⁇ ), alpha synuclein, mutant huntingtin (mHTT), alpha-synuclein, TAR RNA binding protein (TARDBP) and FUS RNA binding protein (FUS).
  • the target protein includes a target protein associated with viral infection.
  • the target proteins related to viral infection include, for example, HBV encoded X protein (HBx), HBV DNA polymerase, HBV capsid glycoprotein, HIV-1 reverse transcriptase, HIV gp120, HCV NS3-4A protease, HCV RNA polymerization Enzyme, HCV envelope protein, EBV DNA polymerase, EBV EBNA1, coronavirus RNA synthase, coronavirus spike protein, coronavirus envelope protein, coronavirus membrane protein, coronavirus nucleocapsid protein, RNA dependence RNA polymerase (RNA-dependent RNA polymerase, RdRp), such as coronavirus RNA-dependent RNA polymerase, Herpesviruses DNA and RNA polymerase, herpesvirus capsid glycoprotein, CMV DNA polymerase, CMV capsidose Protein, RSV membrane protein, RSV capsid protein, RSV RNA polymerase, influenza virus
  • the target protein is a viral protein, such as HBV surface antigen, HBeAg, HBV polymerase protein, HIV Gag protein, HIV Env protein, etc., the function of which is believed to be important for viral replication amplification and function as well as viral diseases. Progress matters.
  • the target protein includes an autoantigen-related target protein.
  • autoantigens associated with autoimmune diseases include islet cell antigen (ICA), insulin (IAA), glutamic acid decarboxylase 65 (GAD65), isletoma antigen-2 (IA-2); and rheumatoid joint RA-related autoantigens (autoantigens) such as citrullinated protein/peptide antibodies, heterogeneous nuclear ribonucleoprotein A2/B1, aldolase, alpha-enolase, calreticulin, heat-activated protein (HSP60), BiP , PGK1, stress-induced phosphoprotein 1, FUSE-BP1/2; autoantigens associated with systemic lupus erythematosus (SLE) such as deoxyribonucleoprotein, SmD1 and SmD3, Clq, sore anticoagulant (LA), cardiolipin (CL), ⁇ 2 glycose (Smith), rheumatoid joint RA-related autoantigens (
  • the disease-causing protein is a target protein associated with a metabolic disease.
  • the target proteins related to metabolic diseases include, for example, target proteins related to atherosclerosis (AS), including but not limited to, CD36, low-density lipoprotein receptor (LDLR), ChemR23 (CMKLR1), mitochondrial dehydrogenase (mitochondrial dehydrogenase) ALDH4A1 (target protein); target proteins for type 2 diabetes, including but not limited to, RalGAP ⁇ 1, dipeptidyl peptidase IV (DPP4); target proteins for non-alcoholic fatty liver disease, including but not limited to , TMEM16A, VAMP3; target proteins for tumor glucose metabolism, including but not limited to, hexokinase (HK), glucose transporter 1 (GLUT1), glucose transporter 4 (GLUT4), Phosphoglycerate dehydrogenase, lactate dehydrogenase; target proteins for tumor lipid metabolism, including but not limited to, ATP citrate lya
  • the targeting domain specifically targets target proteins other than TCR, HLA-I, MICA, and MICB
  • the viral endoplasmic reticulum resident protein is any viral endoplasmic reticulum resident protein described in the present disclosure.
  • the targeting domain specifically targets at least 2 of the following target proteins: TCR, HLA-I, MICA, MICB, and the viral endoplasmic reticulum resident protein is any viral endoplasmic reticulum resident protein described in the present disclosure. Net-resident proteins.
  • the targeting domain specifically targets TCR, HLA-I, MICA, or MICB, and the viral endoplasmic reticulum resident protein is not adenovirus E3-K19.
  • the targeting domain of the chimeric protein construct is connected to the ERAD machinery protein binding domain via a hinge or linker.
  • the hinge comprises the amino acid sequence set forth in SEQ ID NO: 143 (IgG4 hinge) or SEQ ID NO: 141 ((Gly4Ser)2).
  • the chimeric protein constructs provided by the present disclosure may also include protein degradation pathway member binding domains.
  • protein degradation pathway member binding domain refers to any part that can directly or indirectly bind to a member of a protein degradation pathway.
  • a protein degradation pathway can be any pathway that mediates protein degradation within a cell.
  • Known protein degradation pathways include, but are not limited to, the ubiquitination-proteasome pathway, the endosome-lysosome pathway, and the autophagy degradation pathway.
  • ubiquitination-proteasome pathway include, for example, E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, E3 ubiquitin ligases, and the proteasome.
  • E1 ubiquitin-activating enzymes include, for example, UBA1, UBA2, UBA3, UBA5, UBA6, UBA7, ATG7, NAE1, and SAE1.
  • E2 ubiquitin-conjugating enzymes include, e.g., hCdc34, Ubc-Uev1A, UBE2A, UBE2B, UBE2C, UBE2D1, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3, UBE2F, UBE2G1, UBE2G2, UBE2H, UBE2I, UBE2J1, UBE2J 2 , UBE2K, UBE2L3, UBE2L6, UBE2M, UBE2N, UBE2O, UBE2Q1, UBE2Q2, UBE2R1(CDC34), UBE2R2, UBE2S, UBE2T, UBE2U, UBE2V1, UBE2V2, UBE2W, UBE2Z, ATG3, BIRC6, and UFC1.
  • hCdc34 Ubc-Uev1A, UBE2A, UBE2B, U
  • E3 ubiquitin ligases include, for example, von Hippel–Lindau (VHL), Cereblon (CRBN), inhibitor of apoptosis protein (IAP), conch-like ECH-related protein 1 (Kelch-like ECH-associated protein 1, Keap1), RNF4, RNF114, MDM2, LUBAC, FBW7, Met30, HECT, SKP2, beta TRCP1, HUWEI, TRAF6, SMURF1, and E6AP.
  • examples of E3 ubiquitin ligases include, e.g., 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.
  • APC Anaphase-promoting complex
  • UBR5 E
  • Endosome-lysosomal pathway members include, for example, AP-1, AP-2, AP-3, endosome, lysosome, HOPS, ESCRT, GASP, BLOC-1, ESCRT, Retromer, ESCRT, sortingnexin, Dapper2, SNX4, Pincher, Rap1-PDZ-GEF1, clathrin, C3G/CrkL/Shp2/Gab2, etc.
  • autophagy degradation pathway include, for example, chaperone-mediated autophagy (CMA), USP10, G3BP1, ULK1, ATG16L1, TRIM16, FBXO27VDAC, RHOT1, MFN1/2, BNIP3L, FUNDC1, BNIP3, AMBRA1, BCL2LI3, FKBP8, CHDH, DISC1, PHB2, Cardiolipin, SEC62, RTN3, PEX5, PEX14, ABCD3, NUFIP1, Hsc70, etc.
  • CMA chaperone-mediated autophagy
  • the protein degradation pathway member binding domain is a VHL binding domain.
  • the VHL binding domain comprises an amino acid sequence such as SEQ ID NO: 163 (DRHDS(p)GLDS(p)M) or such as SEQ ID NO: 164 (ALAPYIP).
  • the protein degradation pathway member binding domain is a Keap1 binding domain.
  • the Keap1 binding domain comprises an amino acid sequence such as SEQ ID NO: 165 (LDPETGEYL).
  • the protein degradation pathway member binding domain is an E3 ubiquitin ligase binding domain comprising an amino acid sequence such as SEQ ID NO: 166 (DRHDSGLDSM).
  • the protein degradation pathway member binding domain is a CMA binding domain.
  • the CMA binding domain comprises an amino acid sequence such as SEQ ID NO: 167 (KFERQ).
  • the CMA binding domain comprises an amino acid sequence such as SEQ ID NO: 168 (KFERQKILDQRFFE).
  • the protein degradation pathway member binding domain is a proteasome binding domain.
  • the proteasome binding domain is selected from the group consisting of: yeast Rad23 (e.g., S. cerevisiae Rad23), ubiquitin-like (UbL) domain of human Rad23b (hHR23b), HPV E7, anchor Proteasome binding domain of the protein.
  • the proteasome binding domain comprises amino acids 1-77 of yeast Rad23.
  • the proteasome binding domain comprises amino acids 1-83 of human Rad23b.
  • the inventor of the present application found that compared with the basic TPD design (Targeted protein binding domain-Transmembrane domain-ER retention domain, TBD-TMD-ERD), it contains protein degradation pathway member binding domain (ligand for E3 Ligase (E3L), ligand Chimeric protein constructs for E2 Ubiquitin-conjugating enzyme (E2L) or ligand for lysosome (LL)) (e.g., those described above) have significantly improved degradation of target proteins.
  • the functional structure of the viral ER-resident protein has different binding abilities to the ERAD reverse transporter complex.
  • the natural structure also has differences in its interaction with the ER-resident E3ligase, and it also interacts with the normal misfolded protein ERAD in the endoplasmic reticulum. Mechanisms compete for degradation pathways. Therefore, the domains of natural TMD-ERD have differences in their ability to ubiquitylate and degrade target proteins. In order to further improve the degradation of target proteins Efficiency, the introduction of the ubiquitin-proteasome system (UPS) ligand domain and/or the autophagy-lysosome pathway (ALP) ligand domain can further ubiquitinate target proteins retained in the endoplasmic reticulum. Proteasomal degradation or target protein degradation via lysosomes.
  • UPS ubiquitin-proteasome system
  • ALP autophagy-lysosome pathway
  • the targeting domain or ERAD machinery protein binding domain of the chimeric protein construct is connected to the protein degradation pathway member binding domain through a hinge or linker.
  • the hinge comprises the amino acid sequence set forth in SEQ ID NO: 147 (IgG4 hinge) or SEQ ID NO: 144 ((Gly4Ser)2).
  • the present disclosure provides chimeric protein constructs comprising a TCR-targeting protein binding domain and adenovirus E3-19K and one or more protein degradation pathway member binding domains described above. In some embodiments, the present disclosure provides chimeric protein constructs comprising a TCR-targeting protein binding domain and adenovirus E3-19K and E3 ubiquitin ligase binding domains. In some embodiments, the E3 ubiquitin ligase binding domain comprises the amino acid sequence set forth in SEQ ID NO: 162 (DRHDSGLDSMGSGSGALAPYIP).
  • the present disclosure provides chimeric protein constructs comprising a targeting protein binding domain that targets the TCR and a transmembrane domain of a viral endoplasmic reticulum resident protein other than adenovirus E3-19K or a function thereof Variants and endoplasmic reticulum resident domains or functional variants thereof.
  • the viral endoplasmic reticulum resident protein may be at least one selected from the group consisting of: HCMV glycoprotein US2, US11, US3, US10, US6, HSV ICP47, CPXV12, BHV UL49.5, EBV BNFL2a, HCMV UL16, UL141 , UL142, HIV Nef, HIV Vpu, HHV-7U21, HHV-8KK3, HHV-8KK5, MHV-68MK3, HTLV-1p12 and vaccinia virus protein CPXV203.
  • the present disclosure provides chimeric protein constructs comprising a targeting protein binding domain targeting the above target protein (e.g., TCR) and a transmembrane domain of HCMV US2 and/or US11 or a functional variant thereof and endoplasmic reticulum resident domains or functional variants thereof.
  • a targeting protein binding domain targeting the above target protein e.g., TCR
  • a transmembrane domain of HCMV US2 and/or US11 or a functional variant thereof e.g., endoplasmic reticulum resident domains or functional variants thereof.
  • the present disclosure provides chimeric protein constructs comprising a targeting protein binding domain targeting the above-mentioned target protein (e.g., TCR) and the transmembrane domain of HCMV US3 or a functional variant thereof and the endoplasmic reticulum. resident domain or functional variant thereof.
  • a targeting protein binding domain targeting the above-mentioned target protein e.g., TCR
  • the transmembrane domain of HCMV US3 or a functional variant thereof e.g., TCR
  • the present disclosure provides chimeric protein constructs comprising a targeting protein binding domain targeting the above target protein (e.g., TCR) and the transmembrane domain of HCMV US10 or a functional variant thereof and the endoplasmic reticulum. resident domain or functional variant thereof.
  • a targeting protein binding domain targeting the above target protein e.g., TCR
  • the transmembrane domain of HCMV US10 or a functional variant thereof e.g., TCR
  • the present disclosure provides chimeric protein constructs comprising a targeting protein binding domain targeting the target protein (e.g., TCR) described above and HCMV US6, HSV ICP47, CPXV012, EBV BNFL2a, and/or BHV UL49.
  • a targeting protein binding domain targeting the target protein e.g., TCR
  • HCMV US6, HSV ICP47, CPXV012, EBV BNFL2a, and/or BHV UL49 e.g., TCR
  • the present disclosure provides chimeric protein constructs comprising a targeting protein binding domain targeting the above-mentioned target protein (e.g., TCR) and a transmembrane domain of HHV-7US21 or a functional variant thereof and an endosomal Net-resident domain or functional variant thereof.
  • a targeting protein binding domain targeting the above-mentioned target protein e.g., TCR
  • HHV-7US21 or a functional variant thereof e.g., TCR
  • the present disclosure provides chimeric protein constructs comprising a targeting protein binding domain targeting the above-described target protein (e.g., TCR) and a transmembrane domain of HCMV UL16, UL141 and/or UL142 or functions thereof Variants and endoplasmic reticulum resident domains or functional variants thereof.
  • a targeting protein binding domain targeting the above-described target protein e.g., TCR
  • a transmembrane domain of HCMV UL16, UL141 and/or UL142 or functions thereof Variants and endoplasmic reticulum resident domains or functional variants thereof.
  • the present disclosure provides chimeric protein constructs comprising a targeting protein binding domain targeting the target protein (e.g., TCR) described above and HIV Nef, HIV Vpu, HHV-7U21, HHV-8KK3, HHV- The transmembrane domain or functional variant thereof and the endoplasmic reticulum resident domain or functional variant thereof of 8KK5, MHV-68MK3 and/or HTLV-1p12.
  • target protein e.g., TCR
  • the present disclosure provides a chimeric protein construct comprising a targeting protein binding domain targeting the above-described target protein (e.g., TCR) and a transmembrane domain of Cowpox Virus protein CPXV203 or a functional variant thereof and an endogenous Plasma reticulum resident domain or functional variant thereof.
  • a targeting protein binding domain targeting the above-described target protein (e.g., TCR) and a transmembrane domain of Cowpox Virus protein CPXV203 or a functional variant thereof and an endogenous Plasma reticulum resident domain or functional variant thereof.
  • the present disclosure provides chimeric protein constructs comprising a targeting protein binding domain that is not TCR-targeting and adenovirus E3-19K.
  • the chimeric protein constructs described above further comprise one or more protein degradation pathway member binding domains as described above.
  • the chimeric protein constructs provided by the present disclosure are co-expressed with at least one co-expression moiety. Any method suitable for common expression may be used.
  • the chimeric protein construct (or a part thereof) and the at least one co-expression part can be connected through a cleavable linker. When the linker is broken, the chimeric protein construct and the co-expression part can co-express. Express.
  • the chimeric protein construct provided by the present disclosure and at least one co-expression moiety are connected, for example, through a self-cleavable linkage.
  • the self-cleavable linker is a cleavable peptide; e.g., T2A peptide, GSG-T2A peptide, E2A peptide, GSG-E2A peptide, F2A peptide, GSG-F2A peptide, P2A peptide, or GSG- P2A peptide.
  • the T2A includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 144, preferably 85%, 90%, 95%, 96%, 97%, An amino acid sequence with 98% or more than 99% identity, more preferably an amino acid sequence with 98% or more than 99% identity; the amino acid sequence of T2A is shown in SEQ ID NO. 144.
  • the amino acid sequence of the GSG-T2A peptide is as shown in SEQ ID NO. 145.
  • the P2A includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 146, preferably 85%, 90%, 95%, 96%, 97%, An amino acid sequence with 98% or more than 99% identity, more preferably an amino acid sequence with 98% or more than 99% identity; the amino acid sequence of P2A is shown in SEQ ID NO. 146.
  • the amino acid sequence of the GSG-P2A peptide is as shown in SEQ ID NO. 147.
  • the E2A includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 148, preferably 85%, 90%, 95%, 96%, 97%, An amino acid sequence with 98% or more than 99% identity, more preferably an amino acid sequence with 98% or more than 99% identity; the amino acid sequence of E2A is shown in SEQ ID NO. 148.
  • amino acid sequence of the GSG-E2A peptide is shown in SEQ ID NO. 149.
  • the F2A includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 150, preferably 85%, 90%, 95%, 96%, 97%, An amino acid sequence with 98% or more than 99% identity, more preferably an amino acid sequence with 98% or more than 99% identity; the amino acid sequence of F2A is shown in SEQ ID NO. 150.
  • the amino acid sequence of the GSG-F2A peptide is as shown in SEQ ID NO. 151.
  • the co-expression part provided in this application can be any protein or polypeptide with biological functions. Depending on the biological function desired to be achieved, an appropriate co-expression moiety can be selected. For example, to reduce cellular immunogenicity or antigen presentation, proteins capable of degrading or reducing MHC class I or class II molecules can be selected as co-expression moieties. For another example, in order to enable cells to recognize the target protein, a binding domain that recognizes or binds the target protein (such as a chemokine receptor, or a chimeric antigen receptor CAR) can be selected as a co-expression part. For another example, in order to increase immunostimulatory activity, immunostimulatory molecules can be selected as co-expression moieties.
  • examples of the co-expressed portion include, but are not limited to, intact viral ER-resident glycoproteins (e.g., HCMV US2, US3, US11, US10, adenovirus E3-K19, HCMV US6, HSV ICP47), chimeric antigen receptors (CARs), functional T cell receptors (TCRs), chemokine receptors (e.g., CCR4, CCR5, CCR6, CCR7, CCR9, CCR2b, CXCR1, CXCR2, and CXCR4), NK cell activating receptors (e.g., NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, natural cytotoxic receptors, TRAIL, DNAM-1, CD16a, 2B4, NTB-A, CRACC and NKp80), CNK signaling components, cytokines,
  • the co-expressed portion is an intact viral ER-resident glycoprotein, including, but not limited to, HCMV US2, US3, US11, US10, adenovirus E3-K19, HCMV US6, and HSV ICP47.
  • the full-length sequence of the HCMV glycoprotein US2 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 82, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences, preferably, having the activity of degrading MHC class I or class II molecules; so The amino acid sequence of the full-length sequence of the HCMV glycoprotein US2 is shown in SEQ ID NO. 82.
  • the full-length sequence of US2 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 110, preferably 85%, 90%, 95%, 96% , an amino acid sequence with 97%, 98%, or 99% or more identity, more preferably an amino acid sequence with 98% or more than 99% identity, preferably, having the activity of degrading MHC class I or class II molecules; the US2
  • the amino acid sequence of the full-length sequence is shown in SEQ ID NO.110.
  • the full-length sequence of the HCMV glycoprotein US3 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 85, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences, preferably, having the activity of degrading MHC class I or class II molecules; so The amino acid sequence of the full-length sequence of the HCMV glycoprotein US3 is shown in SEQ ID NO.85.
  • the full-length sequence of US3 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 113, preferably 85%, 90%, 95%, 96% , an amino acid sequence with 97%, 98%, or 99% or more identity, more preferably an amino acid sequence with 98% or more than 99% identity, preferably, having the activity of degrading MHC class I or class II molecules; the US3
  • the amino acid sequence of the full-length sequence is shown in SEQ ID NO. 113.
  • the full-length sequence of the HCMV glycoprotein US11 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 88, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences, preferably, having the activity of degrading MHC class I or class II molecules; so The amino acid sequence of the full-length sequence of the HCMV glycoprotein US11 is shown in SEQ ID NO. 88.
  • the full-length sequence of US11 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 116, preferably 85%, 90%, 95%, 96% , an amino acid sequence with an identity of 97%, 98%, or 99% or more, more preferably an amino acid sequence with an identity of 98% or more than 99%, preferably, having the activity of degrading MHC class I or class II molecules; the US11 The amino acid sequence of the full-length sequence is shown in SEQ ID NO. 116.
  • the full-length sequence of the HCMV glycoprotein US10 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 91, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences, preferably, having the activity of degrading MHC class I or class II molecules; so The amino acid sequence of the full-length sequence of the HCMV glycoprotein US10 is shown in SEQ ID NO. 91.
  • the full-length sequence of the adenovirus E3-19K includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 94, preferably 85%, 90%, 95 %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences, preferably, having the activity of degrading MHC class I or class II molecules;
  • the amino acid sequence of the full-length sequence of the adenovirus E3-19K is shown in SEQ ID NO. 94.
  • 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. 100, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences, preferably, having the activity of degrading MHC class I or class II molecules; the HCMV The amino acid sequence of the full-length sequence of US6 is shown in SEQ ID NO. 100.
  • the full-length sequence of HSV ICP47 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 102, preferably 85%, 90%, 95%, 96 %, 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences, preferably, having the activity of degrading MHC class I or class II molecules; the HSV The amino acid sequence of the full-length sequence of ICP47 is shown in SEQ ID NO. 102.
  • the co-expressed moiety is a chimeric antigen receptor (CAR).
  • CAR includes a target protein binding domain (e.g., a tumor-targeting extracellular recognition domain), a transmembrane domain, and an immunoreceptor activation signaling domain (ITAM) (also known as an "intracellular signaling domain" ).
  • ITAM immunoreceptor activation signaling domain
  • the CAR may further comprise a costimulatory domain.
  • the tumor-targeting extracellular recognition domain, transmembrane domain and/or intracellular signaling domain includes a hinge or linker.
  • the tumor-targeting extracellular recognition domain is selected from the group consisting of a tumor antigen-specific binding domain, a tumor microenvironment target antigen-binding domain, and/or a tumor microenvironment-targeting chemotactic receptor.
  • the tumor-targeting extracellular recognition domain is selected from an antibody capable of targeting and recognizing a tumor-associated antigen or a functional fragment thereof, TCR, or a combination thereof; the functional fragment of the antibody is selected from Fd, Fv, Fab, Fab', F(ab')2, Fv(scFv), single chain antibody (scFv) or nanobody, diabody, tribody and quadrubody.
  • the transmembrane domain of the CAR described herein can be derived from any membrane-binding or transmembrane protein, including but not limited to BAFFR, BLAME (SLAMF8), CD2, CD3 ⁇ , CD4, CD5, CD8 , CD9, CD11a (CD18, ITGAL, LFA-l), CD11b, CD11c, CD11d, CD16, CD19, CD22, CD27, CD28, CD29, CD33, CD37, CD40, CD45, CD49a, CD49d, CD49f, CD64, CD80, CD84, CD86, CD96(Tactile), CD100(SEMA4D), CD103, CD134, CD137(4-1BB), CD150(IPO-3, SLAMF1, SLAM), CD154, CD160(BY55), CD162(SELPLG), CD226( DNAM1), CD229(Ly9), CD244(2B4, SLAMF4), CD278(ICOS), CEACAM1, C
  • the transmembrane domain of the CAR is selected from the group consisting of NK cell activating receptor transmembrane domain, DAP10 transmembrane domain, DAP12 transmembrane domain, CD8 transmembrane domain, CD28 transmembrane structure domain, CD4 transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, ICOS transmembrane domain, CTLA-4 transmembrane domain, PD-1 transmembrane domain, LAG-3 transmembrane structure domain, the 2B4 transmembrane domain and the BTLA transmembrane domain, and combinations thereof.
  • the transmembrane domain of the CAR is selected from the group consisting of CD8 transmembrane domain, ⁇ and/or ⁇ chain transmembrane domain of T cell receptor, CD28 transmembrane domain, CD3 epsilon transmembrane structure domain, CD45 transmembrane domain, CD4 transmembrane domain, CD5 transmembrane domain, CD8 transmembrane domain, CD9 transmembrane domain, CD16 transmembrane domain, CD22 transmembrane domain, CD33 transmembrane domain, CD37 transmembrane domain, CD64 transmembrane domain, CD80 transmembrane domain, CD86 transmembrane domain, CD134 transmembrane domain, CD137 transmembrane domain, CD154 transmembrane domain, GITR transmembrane domain and combinations thereof .
  • the immune receptor activation signaling domain is from an intracellular activation signaling domain of an immune receptor; preferably, the immune receptor is selected from TCR ⁇ , CD2, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, FcR ⁇ , CD66d, Fc ⁇ RI, Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIII, Dectin-1, CLEC-1, CD72, CD79A, CD79B;
  • the immune receptor activation signaling domain (ITAM ) is fused to an NK cell signal transducer or a functional variant thereof; preferably, the immune receptor is CD3 ⁇ .
  • the immunoreceptor activation signaling domain is derived from CD3 ⁇ , common FcR ⁇ (FCER1G), Fc ⁇ RIIa, FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), Fc ⁇ RI CD66d, DAP10 and DAP12 and other intracellular signaling domains.
  • the intracellular signaling domain of the CAR includes the intracellular signaling domain and/or the costimulatory signaling domain of an NK cell activating receptor.
  • the T cell costimulatory signaling domain is derived from the intracellular signaling domain of a costimulatory molecule; preferably, the costimulatory molecule is selected from the group consisting of MHC class I molecules, TNF receptor proteins, immune Globulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD16, CD27, CD28, CD30, CD40, CD38, CD35, CD79A, CD79B, CDS, ICAM-1, LFA-1, (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD
  • the costimulatory signaling domain can be selected from the group consisting of NKG2D intracellular signaling domain, DAP10 intracellular signaling domain, DAP12 intracellular signaling domain, NCR intracellular signaling domain, CD28 intracellular signaling domain, 4-1BB intracellular signaling domain, OX40 intracellular signaling domain, and ICOS intracellular signaling domain.
  • the hinge and/or transmembrane domains of a CAR described herein provide cell surface presentation of the extracellular domain of the CAR.
  • the hinge of the CAR described herein can be derived from any membrane-bound or transmembrane protein, including but not limited to BAFFR, BLAME (SLAMF8), CD2, CD3 ⁇ , CD4, CD5, CD8, CD9, CD11a (CD18, ITGAL, LFA- l), CD11b, CD11c, CD11d, CD16, CD19, CD22, CD27, CD28, CD29, CD33, CD37, CD40, CD45, CD49a, CD49d, CD49f, CD64, CD80, CD84, CD86, CD96(Tactile), CD100( SEMA4D), CD103, CD134, CD137(4-1BB), CD150(IPO-3, SLAMF1, SLAM), CD154, CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(Ly
  • the linker is a flexible linker; preferably, the flexible linker comprises the amino acid sequence shown (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 x and y are not both 0; more preferably, the linker includes the amino acid sequence shown in SEQ ID NO.140 or the amino acid sequence shown in SEQ ID NO.141 sequence.
  • the hinge is an IgG1 hinge or an IgG4 hinge.
  • the IgG1 hinge comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 142, preferably 85%, 90%, 95%, 96%, 97% , an amino acid sequence with 98% or more than 99% identity, and more preferably an amino acid sequence with 98% or more than 99% identity; the amino acid sequence of the IgG1 hinge is shown in SEQ ID NO. 142.
  • the IgG4 hinge comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 143, preferably 85%, 90%, 95%, 96%, 97% , an amino acid sequence with 98% or more than 99% identity, and more preferably an amino acid sequence with 98% or more than 99% identity; the amino acid sequence of the IgG4 hinge is shown in SEQ ID NO. 143.
  • the co-expressed moiety is a CNK receptor.
  • the CNK receptor includes a chimeric NK activating receptor component and a chimeric NK signal transduction component (also referred to as a "CNK signal transduction component").
  • the chimeric NK activating receptor component may at least comprise an NK cell activating receptor or a functional variant thereof.
  • the NK cell activating receptor comprises: (a) an NK cell activating receptor extracellular domain (ED) or a functional variant thereof, (b) an NK cell activating receptor transmembrane domain (TMD) ) or a functional variant thereof, and (c) an NK cell activating receptor intracellular domain (ICD) or a functional variant thereof; optionally, the NK cell activating receptor extracellular domain or a functional variant thereof, A hinge or joint is included between the NK cell activating receptor transmembrane domain or its functional variant and/or the NK cell activating receptor intracellular domain or its functional variant.
  • ED NK cell activating receptor extracellular domain
  • TMD NK cell activating receptor transmembrane domain
  • ICD NK cell activating receptor intracellular domain
  • a hinge or joint is included between the NK cell activating receptor transmembrane domain or its functional variant and/or the NK cell activating receptor intracellular domain or its functional variant.
  • the NK cell activation receptor is selected from NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, natural cytotoxicity receptors (NCR), TRAIL, DNAM-1, Signaling lymphocytic activation molecule (SLAM) family molecule 2B4 (also known as CD244), DNAX attachment molecule 1 (DNAM-1, also known as CD226), CD16a, 2B4, NTB-A, CRACC (CS1) and NKp80;
  • the natural cytotoxic receptors include NKp46 (also known as NCR1 or CD335), NKp44 (also known as NCR2 or CD336) and NKp30 (also known as NCR3 or CD337).
  • the natural cytotoxic receptor is selected from NKp46, NKp44, and NKp30.
  • the NK cell activating receptor is an NK cell activating receptor of mammalian origin; preferably, the mammal is selected from the group consisting of human, primate, mouse, horse, cow, sheep, Goats, cats, pigs, dogs, llamas, alpacas, elephants, squirrels, guinea pigs.
  • the NK cell activating receptor is a recombinant NK cell activating receptor comprising NK cell activating receptor domains from different sources.
  • the NK cell activating receptor is a human NK cell activating receptor; preferably, the NK cell activating receptor is a recombinant comprising different human NK cell activating receptor domains. NK cell activating receptor.
  • the NK cell activating receptor is a murine NK cell activating receptor; preferably, the NK cell activating receptor is a recombinant comprising different murine NK cell activating receptor domains. NK cell activating receptor.
  • the NK cell activating receptor is a recombinant NK cell activating receptor comprising human and murine NK cell activating receptor domains.
  • the extracellular domain of the NK cell activating receptor is the extracellular domain of a human or murine NK cell activating receptor.
  • the transmembrane domain of the NK cell activating receptor is that of a human or murine NK cell activating receptor.
  • the intracellular domain of the NK cell activating receptor is the intracellular domain of a human or murine NK cell activating receptor.
  • the functional variant of the NK cell activating receptor is selected from a mutant of the NK cell activating receptor, a wild-type fusion protein, or a fusion protein of a wild-type and a mutant type.
  • 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. 37, preferably 85%, 90%, 95%, 96%, 97 %, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the extracellular domain of human NKG2D is shown in SEQ ID NO. 37.
  • the full-length sequence of human NKG2D includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 38, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human NKG2D is shown in SEQ ID NO. 38.
  • 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. 39, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the extracellular domain of mouse NKG2D is shown in SEQ ID NO. 39.
  • 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: 40, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the full-length amino acid sequence of mouse NKG2D is shown in SEQ ID NO. 40.
  • 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: 41, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of human and mouse recombinant NKG2D is as shown in SEQ ID NO.41 Show.
  • the full-length sequence of human NKG2C includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 42, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human NKG2C is shown in SEQ ID NO. 42.
  • 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: 43, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human NKG2E is shown in SEQ ID NO. 43.
  • 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: 44, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human NKG2F is shown in SEQ ID NO. 44.
  • the full-length sequence of human CD94 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 45, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably amino acid sequences with 98% or 99% or more identity; the amino acid sequence is as shown in SEQ ID NO. 45.
  • the full-length sequence of human KIR2DL4 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 46, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human KIR2DL4 is shown in SEQ ID NO. 46.
  • the full-length sequence of human KIR2DS1 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 47, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human KIR2DS1 is shown in SEQ ID NO. 47.
  • 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: 48, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human KIR2DS2 is shown in SEQ ID NO. 48.
  • the full-length sequence of human KIR2DS4 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 49, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human KIR2DS4 is shown in SEQ ID NO. 49.
  • the full-length sequence of human KIR3DS1 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 50, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human KIR3DS1 is shown in SEQ ID NO. 50.
  • the full-length sequence of human NKp46 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 51, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human NKp46 is shown in SEQ ID NO. 51.
  • the full-length sequence of human NKp44 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 52, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human NKp44 is shown in SEQ ID NO. 52.
  • the full-length sequence of human NKp30 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 53, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human NKp30 is shown in SEQ ID NO. 53.
  • the full-length sequence of human DNAM1 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 54, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human DNAM1 is shown in SEQ ID NO. 54.
  • the full-length sequence of human TRAIL contains 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 55.
  • a homogeneous amino acid sequence is preferably an amino acid sequence with an identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more, and more preferably an amino acid sequence with an identity of 98% or 99% or more;
  • the amino acid sequence of the full-length sequence of human TRAIL is shown in SEQ ID NO. 55.
  • 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: 56, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human CD16a is shown in SEQ ID NO. 56.
  • the full-length sequence of human 2B4 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 57, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human 2B4 is shown in SEQ ID NO. 57.
  • the full-length sequence of human NTB-A includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 58, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the full-length sequence of human NTB-A is as shown in SEQ ID NO.58 Show.
  • 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: 59, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human CRACC is shown in SEQ ID NO. 59.
  • the full-length sequence of human NKp80 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 60, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human NKp80 is shown in SEQ ID NO. 60.
  • the CNK signaling component comprises at least (i) an NK cell signal adapter (eg, DAP10 or DAP12) or a functional variant thereof.
  • the NK cell signal transducer comprises: (a) an NK cell signal transducer extracellular domain (ED) or a functional variant thereof, (b) an NK cell signal transducer transmembrane domain (TMD) ) or a functional variant thereof, and (c) an NK cell signal transducer intracellular domain (ICD) or a functional variant thereof; optionally, the NK cell signal transducer extracellular domain or a functional variant thereof, A hinge or joint is included between the NK cell signal transducer transmembrane domain or a functional variant thereof and/or the NK cell signal transducer intracellular domain or a functional variant thereof.
  • the NK cell signal transducer is a mammalian-derived NK cell signal transducer; preferably, the mammal is selected from the group consisting of human, primate, mouse, horse, cow, sheep, Goats, cats, pigs, dogs, llamas, alpacas, elephants, squirrels, guinea pigs.
  • the NK cell signal transducer is a recombinant NK cell signal transducer comprising NK cell signal transducer domains from different sources.
  • the NK cell signal transducer is a human NK cell signal transducer; preferably, the NK cell signal transducer is a recombinant comprising different human NK cell signal transducer domains. NK cell signal transducer.
  • the NK cell signal transducer is a murine NK cell signal transducer; preferably, the NK cell signal transducer is a recombinant comprising different murine NK cell signal transducer domains. NK cell signal transducer.
  • the NK cell signal transducer is a recombinant NK cell signal transducer comprising human and murine NK cell signal transducer domains.
  • the extracellular domain of the NK cell signal transducer is the extracellular domain of a human or murine NK cell signal transducer.
  • the transmembrane domain of the NK cell signal transducer is a transmembrane domain of a human or murine NK cell signal transducer.
  • the intracellular domain of the NK cell signal transducer is an intracellular domain of a human or murine NK cell signal transducer
  • the NK cell signal converter in the CNK signal switching component is DAP10 or DAP12.
  • the CNK cell signal transducer functional variant is selected from a mutant of DAP10 or DAP12, or a fusion protein of DAP10 and DAP12, or a fusion protein of wild-type DAP10 or DAP12 and mutant DAP10 or DAP12 .
  • the CNK signaling component further comprises (ii) an immunoreceptor activation signaling domain (ITAM) and/or (iii) a T cell costimulatory signaling domain.
  • ITAM immunoreceptor activation signaling domain
  • the NK cell signal transducer or functional variant thereof, the immunoreceptor activation signaling domain (ITAM) and/or the T cell costimulatory signaling domain comprise Hinge or linker;
  • the NK cell signal transducer or functional variant thereof is fused to the immune receptor activation signaling domain (ITAM) domain.
  • the immune receptor activation signaling domain is from an intracellular activation signaling domain of an immune receptor; preferably, the immune receptor is selected from TCR ⁇ , CD2, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, FcR ⁇ , CD66d, Fc ⁇ RI, Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIII, Dectin-1, CLEC-1, CD72, CD79A, CD79B;
  • the immune receptor activation signaling domain (ITAM ) is fused to an NK cell signal transducer or a functional variant thereof; preferably, the immune receptor is CD3 ⁇ .
  • the T cell costimulatory signaling domain is derived from the intracellular signaling domain of a costimulatory molecule.
  • 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. 61, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably with An amino acid sequence with an identity of 98% or above; the amino acid sequence of the full-length sequence of human DAP10 is shown in SEQ ID NO. 61.
  • the full-length sequence of human DAP10 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 62, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human DAP10 is shown in SEQ ID NO. 62.
  • 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. 63, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the transmembrane domain of human DAP10 is shown in SEQ ID NO. 63.
  • the full-length sequence of human DAP12 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 64, preferably 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, or 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequence of the full-length sequence of human DAP12 is shown in SEQ ID NO. 64.
  • 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. 65, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the transmembrane domain of human DAP12 is shown in SEQ ID NO. 65.
  • 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. 66, preferably 85%, 90%, Amino acid sequences with 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or 99% or more identity; the amino acid sequences of the transmembrane domains of human DAP10 and human DAP12 are as follows SEQ ID NO.66 is shown.
  • 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. 67, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP10-DAP12 fusion protein sequence is shown in SEQ ID NO. 67.
  • the human CD3zeta intracellular signaling domain sequence includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 68, preferably 85%, 90%, 95%, Amino acid sequences with 96%, 97%, 98%, or 99% or more identity, more preferably amino acid sequences with 98% or 99% or more identity; the amino acid sequence of the human CD3zeta intracellular signaling domain sequence is such as SEQ ID NO. 68 shown.
  • 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. 69, preferably 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP10-CD3zeta sequence is shown in SEQ ID NO. 69.
  • 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. 70, preferably 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP12-CD3zeta sequence is shown in SEQ ID NO. 70.
  • 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. 71, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP10-DAP12-CD3zeta sequence is shown in SEQ ID NO. 71.
  • 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. 72, preferably 85%, 90%, 95%, Amino acid sequences with 96%, 97%, 98%, or 99% or more identity, more preferably amino acid sequences with 98% or 99% or more identity; the amino acid sequence of the human 41BB intracellular signaling domain sequence is such as SEQ ID NO. 72 shown.
  • 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. 73, preferably 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP10-41BB sequence is shown in SEQ ID NO. 73.
  • 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. 74, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP10-41BB-CD3zeta sequence is shown in SEQ ID NO. 74.
  • 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. 75, preferably 85%, 90%, 95%, Amino acid sequences with 96%, 97%, 98%, or 99% or more identity, more preferably amino acid sequences with 98% or 99% or more identity; the amino acid sequence of the human CD28 intracellular signaling domain sequence is such as SEQ ID NO. 75 shown.
  • 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. 76, preferably 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP10-CD28 sequence is shown in SEQ ID NO. 76.
  • 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. 77, preferably 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, better Select an amino acid sequence with an identity of 98% or more than 99%; the amino acid sequence of the human DAP10-CD28-CD3zeta sequence is shown in SEQ ID NO. 77.
  • 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. 78, preferably 85%, 90%, 95%, 96%, 97 %, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP12-41BB sequence is shown in SEQ ID NO. 78.
  • 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. 79, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP12-41BB-CD3zeta sequence is shown in SEQ ID NO. 79.
  • 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. 80, preferably 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP12-CD28 sequence is shown in SEQ ID NO. 80.
  • 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. 81, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the human DAP12-CD28-CD3zeta sequence is shown in SEQ ID NO. 81.
  • the CNK signal transfer component comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 61-81.
  • the chimeric protein construct, the NK cell activating receptor and the CNK signal transduction component form a multifunctional complex, wherein the multifunctional complex includes as shown in SEQ ID NO. 152 Amino acid sequence.
  • the multifunctional complex includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 152, preferably 85%, 90%, 95%, 96% , 97%, 98%, or 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; the amino acid sequence of the multifunctional complex includes as shown in SEQ ID NO. 152 TCR antibody single chain antibody.
  • the disclosure also provides a chimeric nucleic acid construct encoding any of the chimeric protein constructs described in the application.
  • the nucleic acid molecules may include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), including those with ⁇ - LNA with D-ribose configuration, ⁇ -LNA with ⁇ -L-ribose configuration (diastereomer of LNA), 2'-amino-LNA with 2'-amino functionalization and 2'-amino-LNA with 2'- Amino-functionalized 2'-amino- ⁇ -LNA), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and/or chimeras and/or combinations thereof.
  • Exemplary DNA includes, but is not limited to, plasmid DNA (pDNA)) and the like.
  • Exemplary RNAs include, but are not limited to, mRNA, circular
  • the nucleic acid molecule is mRNA.
  • mRNA is any RNA that encodes at least one protein, and the encoded protein can be produced in vitro, in vivo, in situ, or ex vivo.
  • the nucleic acid sequences set forth in this application may enumerate "T” among representative DNA sequences, but where the sequence represents RNA (e.g., mRNA), "T” will Replace it with "U”.
  • any DNA disclosed and identified herein by a specific sequence identification number also discloses a corresponding RNA (eg, mRNA) sequence that is complementary to the DNA, wherein each "T" of the DNA sequence is replaced by a "U”.
  • the nucleic acid molecule comprises a nucleotide sequence having 80% or more identity with the nucleotide sequence shown in SEQ ID NO. 154, preferably 85%, 90%, 95%, A nucleotide sequence having an identity of 96%, 97%, 98%, or 99% or more, and a nucleotide sequence having an identity of 98% or 99% or more is more preferred.
  • the nucleic acid molecule is mRNA synthesized in vitro.
  • the in vitro synthesized mRNA has modifications, optionally, including one or more modifications selected from the group consisting of: 5'UTR, 5'UTR, polyA tail, 5'-added cap, and one or more modified nucleotides in the coding region.
  • nucleic acid molecules e.g. mRNA
  • Naturally occurring eukaryotic mRNA molecules may contain stabilizing elements including, but not limited to, an untranslated region (UTR) at its 5'-end (5'UTR) and/or a UTR at its 3'-end (3'UTR ), as well as other structural features such as 5'-cap structure and 3'-poly A tail.
  • UTR untranslated region
  • 3'UTR 3'-end
  • 5'UTR and 3'UTR are usually transcribed from genomic DNA and are part of premature mRNA. Characteristic structures of mature mRNA such as 5'-cap structure and 3'-polyA tail are often added to transcribed (premature) mRNA during mRNA processing.
  • 5'UTR refers to the region of an mRNA located directly upstream (i.e., 5') of the initiation codon (i.e., the first codon of the ribosome-translated mRNA transcript), which does not encode a polypeptide.
  • the 5'UTR may contain a promoter sequence. Such promoter sequences are known in the art.
  • 3'UTR refers to the region of an mRNA immediately downstream (ie, 3') of a stop codon (ie, the codon of the mRNA transcript indicating termination of translation), which does not encode a polypeptide.
  • a “poly-A tail” is a downstream region of an mRNA, e.g., directly downstream (i.e., 3') of the 3'UTR, that contains multiple consecutive adenosine monophosphates.
  • a polyA tail may contain from 10 to 300 adenosine monophosphates.
  • a poly A tail might 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 monophosphate.
  • the polyA tail contains 50 to 250 adenosine monophosphate.
  • the poly-A tail functions to protect the mRNA from enzymatic degradation (e.g., in the cytoplasm) and facilitates transcription termination and/or export and translation of the mRNA from the nucleus.
  • the 3'-polyA tail is usually a stretch of adenine nucleotides added to the 3'-end of transcribed mRNA. In some cases, it can contain up to about 400 adenine nucleotides. The length of the 3'-polyA tail can affect the stability of the mRNA molecule itself.
  • 5'-capping of polynucleotides can be accomplished simultaneously during the in vitro transcription reaction by using the following chemical RNA cap analog to generate a 5'-guanosine cap structure according to the manufacturer's protocol: 3'-O-Me-m7G( 5')ppp(5')G[ARCA cap], G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G (New England Biolabs, Ipswich, MA).
  • 5'-capping of modified RNA can be accomplished post-transcriptionally using the vaccinia virus capping enzyme to generate the "Cap 0" structure: m7G(5')ppp(5')G (New England Biolabs, MA Puswich).
  • the Cap 1 structure can be generated using vaccinia virus capping enzyme and 2'-0 methyltransferase: m7G(5')ppp(5')G-2'-O-methyl.
  • the Cap 2 structure can be generated from the Cap 1 structure, followed by 2'-O-methylation of the 5'-antepenultimate nucleotide using a 2'-O methyltransferase.
  • the Cap 3 structure can be generated from the Cap 2 structure, followed by 2'-O-methylation of the 5'-preantepenultimate nucleotide using a 2'-O methyltransferase.
  • Enzymes can be derived from recombinant sources.
  • the mRNA molecule containing at least one of the above-mentioned stabilizing elements can significantly increase the expression level of the protein encoded by it, and when two or more of the above-mentioned stabilizing elements are used, the protein expression level is compared with that of the mRNA using a single stabilizing element. Show synergy.
  • the mRNA described in the present disclosure has one or more AU-rich sequences removed.
  • AU-rich sequences also known as AURES, are destabilizing sequences found in the 3'UTR.
  • the mRNA of the present disclosure further comprises an open reading frame (ORF) encoding a signal peptide.
  • the signal peptide may comprise amino acids 15-60 of the N-terminus of the protein, which are usually required for membrane transport across the secretory pathway and, therefore, generally control the entry of most proteins into the secretory pathway in eukaryotes and prokaryotes.
  • the length of the signal peptide may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 amino acids.
  • An "open reading frame” is a contiguous stretch of DNA that begins with a start codon (eg, methionine (ATG)) and ends with a stop codon (eg, TAA, TAG, or TGA) and encodes a polypeptide.
  • a start codon eg, methionine (ATG)
  • a stop codon eg, TAA, TAG, or TGA
  • the signal peptide can include one of the following sequences: MDSKGSSQKGSRLLLLLVVSSNLLLPQGVVG (SEQ ID NO:155), MDWTWILFLVAAATRVHS (SEQ ID NO:156), METPAQLLFLLLLLWLPDTTG (SEQ ID NO:157), MLGSNSGQRVVFTILLVAPAYS (SEQ ID NO:158) , MKCLLYLAFLFIGVNCA (SEQ ID NO: 159), MWLVSLAIVTACAGA (SEQ ID NO: 160) and MFVFLVLLPLVSSQC (SEQ ID NO: 161).
  • nucleic acid molecules provided in this application can be synthesized through solid-phase technology synthesis, liquid-phase chemical synthesis, enzymatic ligation, and combinations thereof. Synthesized nucleic acid molecules can be purified and quantified. For details, see WO2021222304, the entire content of which is incorporated herein by reference.
  • the present disclosure also provides a vector comprising a nucleic acid molecule as provided by the present disclosure operably linked to at least one polynucleotide regulatory element (eg, a promoter) to express the The chimeric protein construct encoded by the nucleic acid molecule.
  • a polynucleotide regulatory element eg, a promoter
  • the vector is selected from plasmids, nanoplasmids, cosmids, viral vectors, minicircles, RNA vectors, or linear or circular DNA (eg, transposon DNA) or RNA molecules.
  • the viral vector is selected from the group consisting of retroviruses, lentiviral vectors, adenoviruses, parvoviruses (e.g., adeno-associated viruses), adeno-associated virus (AAV) vectors, coronaviruses, negative-strand RNA viruses such as Myxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., Myxoma and Sendai), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses, including adenovirus, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia virus, fowlpox virus, and canary
  • the viral vector is a retroviral vector.
  • the retrovirus is selected from the group consisting of avian leukocyte hyperplasia-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV collection, lentivirus, foamy virus.
  • the viral vector is a lentiviral vector. In some embodiments, the viral vector is an oncolytic viral vector.
  • Oncolytic virus viral vectors refer to viral vectors based on oncolytic viruses, into which exogenous nucleic acid sequences can be inserted.
  • the lentiviral vector is selected from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or ovine demyelinating leukoencephalitis lentivirus.
  • the vector is a transposon-based expression vector.
  • Transposons are DNA sequences that can change their position within the genome.
  • the nucleic acid molecule encoding the chimeric protein constructs provided herein is flanked by terminal repeats recognized by transposase enzymes that mediate transposon movement.
  • the transposase can be co-delivered as a protein, encoded on the same vector as the chimeric protein construct, or encoded on a separate vector.
  • Non-limiting examples of transposon systems include Sleeping Beauty, Piggyback, Frog Prince, and Prince Charming.
  • vectors provided by the present disclosure can be combined with other vectors comprising other nucleic acid molecules encoding at least one co-expression moiety.
  • the vector further comprises a promoter; preferably, the promoter is an EF1 ⁇ promoter or a CMV promoter.
  • the chimeric protein construct provided by the present disclosure can be in the same vector and the same promoter as the co-expression part, or not Control expression under the same promoter, or express in multiple vectors.
  • the present disclosure also provides an engineered cell that expresses a chimeric protein construct as provided by the present disclosure, or contains a chimeric nucleic acid construct or vector as provided by the present disclosure.
  • the engineered cells simultaneously express chimeric protein constructs as provided by the present disclosure and co-expression moieties as described above.
  • the co-expression portions and the targeting protein binding domains of the chimeric protein constructs can be combined to achieve specific purposes.
  • the co-expressed portion can be a protein that further inhibits immune rejection (e.g., the ER-resident glycoprotein of the virus etc.), proteins targeting the tumor microenvironment (e.g., chemokine receptors, etc.), proteins that stimulate immune activity (e.g., cytokines, immune cell germline proteins CD7, CD5, etc.), the resulting cells are It will have reduced immunogenicity (or immune rejection) and/or enhanced immune activity when introduced into the human body. This is very useful in cellular immunotherapy and other therapeutic cell transplants.
  • the cells may be immune cells (eg, T cells), stem cells, kidney cells, pancreatic islet cells, cardiomyocyte
  • the cells are immune cells selected from: T cells, natural killer (NK) cells, B cells, macrophages, monocytes, dendritic cells, neutrophils, or ⁇ T cells.
  • the T cells can be selected from the group consisting of: CD8+ T cells, CD4+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, regulatory T cells, natural killer T cells, ⁇ - ⁇ T cells, cytokine-induced killer (CIK) T cells, and tumor-infiltrating lymphocytes.
  • the engineered cells are T cells.
  • the T cells may further express the ability to specifically target Receptors for tumor-related markers, including but not limited to CAR, TCR, NK cell activating receptor components, etc.
  • the engineered cells are T cells that simultaneously express a chimeric protein construct as provided by the present disclosure and one or more selected from: adenovirus E3-K19, CAR, NK cells Activating receptors and CNK signaling components.
  • the engineered cells are T cells that simultaneously express a chimeric protein construct as provided in this disclosure and an NK cell activating receptor and optionally a CNK signaling component.
  • the engineered cells are T cells that simultaneously express a chimeric protein construct as provided by this disclosure, adenovirus E3-K19, and a CAR.
  • the present disclosure also provides a method of producing an engineered cell (e.g., a T cell) as described herein, comprising converting a nucleic acid molecule as described herein under conditions suitable for expression of a nucleic acid molecule as described herein.
  • the vector is introduced into the starting cells.
  • CAR-T cells Many methods for generating CAR-T cells known in the art can also be applied to generate engineered cells as described herein. For example, methods for generating CAR-T cells are described in Zhang et al., Engineering CAR-T cells, Biomarker Research (2017) 5:22. The methods provided herein can include one or more steps selected from: obtaining a starting cell, culturing (including expanding, optionally including activating) the starting cell, and genetically modifying the cell.
  • the starting cell may be a stem cell, which may be a hematopoietic progenitor cell (eg, T cell progenitor cell, NK cell progenitor cell, macrophage progenitor cell), hematopoietic stem cell (HSC), CD34+ cell, embryonic cell line stem cell, mesenchymal stem cell, 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 (eg, T cells) may be isolated from a subject (eg, a human subject).
  • immune cells are obtained from a subject of interest, such as a subject suspected of having a particular disease or disorder, a subject suspected of being susceptible to a particular disease or disorder, about to experience, currently experiencing Subjects who have received treatment for a specific disease or condition can also be healthy volunteers or healthy donors; immune cells can also come from blood banks.
  • the immune cells may be autologous or allogeneic to the subject of interest.
  • the immune cells can be collected from any location in the subject where they are present, including, but not limited to, blood, umbilical cord blood, spleen, thymus, lymph nodes, pleural effusion, splenic tissue, tumors, and bone marrow. Isolated immune cells can be used directly or stored for a period of time, such as frozen.
  • immune cells are activated and expanded simultaneously with, before, or/and after genetic modification.
  • immune cells are activated and/or expanded in vitro, ex vivo, or in vivo. Methods of activating and expanding immune cells have been described in the art and can be used in the methods described herein.
  • T cells can be activated and expanded by surface contact with attached agents that stimulate signals associated with the CD3/TCR complex and ligands that stimulate costimulatory molecules on the surface of the T cells.
  • a population of T cells can be stimulated, such as by contact with an anti-CD3 antibody or antigen-binding fragment thereof or an anti-CD2 antibody immobilized on a surface, or with a protein kinase C activator (eg, bryostatin) and a calcium ionophore.
  • a protein kinase C activator eg, bryostatin
  • a calcium ionophore e.g, calcium ionophore.
  • ligands that bind to the accessory molecules are used.
  • a population of T cells can be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation.
  • anti-CD3 antibodies and anti-CD28 antibodies can be used.
  • primary stimulatory signals and costimulatory signals for T cells can be provided by different protocols.
  • nucleic acid molecules provided herein are introduced into the starting cell using retroviral vectors (eg, lentiviral vectors).
  • retroviral vectors eg, lentiviral vectors
  • the nucleic acid molecules provided herein can be cloned into a lentiviral vector and expression can be driven from its endogenous promoter, the lentiviral long terminal repeat, or a promoter specific for the target cell type of interest.
  • Common delivery methods used to deliver viral vectors include, but are not limited to, electroporation, microinjection, gene gun, and magnetofection.
  • Genetically modified cells can also be achieved by using LNP to deliver nucleic acid molecules.
  • Nucleic acid molecules include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion.
  • Transposase or targeting nucleases e.g. zinc finger nucleases, meganucleases or TALE nucleases, CRISPR
  • CRISPR CRISPR
  • engineered cells provided herein are prepared by transfecting a nucleic acid molecule encoding a chimeric protein construct provided herein into a starting cell prior to administration.
  • engineered cells provided herein can 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 reduced expression of immunogenic molecules (e.g., TCR, HLA, etc.) and/or expression of immunosuppressive molecules (e.g., PD-1, etc.) on the cell surface and/or enhanced tumor targeting.
  • Receptor such as CAR, engineered TCR, CNK receptor, etc. expression.
  • the present disclosure also provides a population of cells produced ex vivo by the above method.
  • at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cell population express detectable levels of an expression marker polypeptide provided herein (e.g., with The chimeric protein construct described above can cleave linked EGFR truncate (EGFRt)).
  • the expression level of the EGFR truncated form may be representative of the expression level of the chimeric protein construct described herein in a cell population.
  • the present disclosure also provides a pharmaceutical composition or a kit comprising (i) a chimeric protein construct, nucleic acid molecule, vector or cell population as described herein, and (ii) a pharmaceutical acceptable medium.
  • pharmaceutically acceptable medium refers to any and all solvents, dispersion media, coatings, antibacterial agents, or other solvents, dispersion media, coatings, antibacterial agents, and/or solvents that facilitate storage and administration of the chimeric protein constructs, nucleic acid molecules, expression vectors, and/or cell populations provided by the present disclosure.
  • the pharmaceutically acceptable vehicle may include any suitable component, such as, but not limited to, saline, liposomes, polymeric excipients, colloids, or carrier particles.
  • a pharmaceutically acceptable vehicle is a saline that can dissolve or disperse the chimeric protein constructs, nucleic acid molecules, expression vectors, and/or cell populations provided by the present disclosure.
  • saline include, but are not limited to, buffered saline, sucrose solution, physiological saline, acetate buffer, phosphate buffer, citrate buffer, bicarbonate buffer, saline solution, and polysorbate solution.
  • the pharmaceutically acceptable vehicle is liposomes.
  • Liposomes are unilamellar or multilamellar vesicles with a membrane formed of lipophilic material and an internal aqueous portion. Nucleic acid molecules and/or vectors provided by the present disclosure can be encapsulated in the aqueous portion of liposomes.
  • Exemplary liposomes include, but are not limited to, liposomes based on 3[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chlo), liposomes based on N-(2,3 -Liposomes based on dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA), and 1,2-dioleoyloxy-3-trimethylpropane (DOTAP ) liposomes.
  • DC-Chlo 3[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol
  • DOTMA dioleoyloxypropyl-N,N,N-trimethylammonium chloride
  • DOTAP 1,2-dioleoyloxy-3-trimethylpropane
  • the pharmaceutically acceptable vehicle is a polymeric excipient including, but not limited to, microspheres, microcapsules, polymeric micelles, and dendrimers.
  • Nucleic acid molecules and/or vectors provided by the present disclosure can be encapsulated, adhered, or coated on polymer-based components by methods known in the art (see, e.g., W. Heiser, Nonviral gene transfer techniques, published by Humana Press, 2004; U.S. patent 6025337; Advanced Drug Delivery Reviews, 57(15):2177-2202(2005)).
  • the pharmaceutically acceptable vehicle is a colloid or carrier particle, such as gold colloids, gold nanoparticles, silica nanoparticles, and multi-segmented nanorods.
  • Nucleic acid molecules and/or vectors provided by the present disclosure may be coated with, adhered to, or combined with the vector in any suitable manner known in the art (see, e.g., M. Sullivan et al., Gene Therapy, 10:1882 –1890(2003),C.Mcclntosh 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)).
  • compositions may further include additives including, but not limited to, stabilizers, preservatives, and transfection enhancers that aid cellular uptake of the drug.
  • stabilizers may include, but are not limited to, sodium glutamate, glycine, EDTA, and albumin.
  • preservatives may include, but are not limited to, 2-phenoxyethanol, sodium benzoate, potassium sorbate, methyl hydroxybenzoate, phenols, thimerosal, and antibiotics.
  • Suitable transfection promoters may include, but are not limited to, calcium ions.
  • compositions provided herein may be administered by any suitable route known in the art, including, but not limited to, parenteral, oral, enteral, buccal, nasal, topical, rectal, vaginal, intramuscular, intranasal, transmucosal , epidermal, transdermal, dermal, ocular, pulmonary and subcutaneous routes of administration.
  • the pharmaceutical compositions provided herein can be administered to a subject in a formulation or formulation suitable for each route of administration.
  • Formulations suitable for administration of pharmaceutical compositions may include, but are not limited to, solutions, dispersions, emulsions, powders, suspensions, aerosols, sprays, nasal drops, liposome-based formulations, patches, implants and suppositories.
  • formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. Methods of preparing these formulations or pharmaceutical compositions include the steps of providing the nucleic acid molecules of the present disclosure to one or more pharmaceutically acceptable vehicles and optionally one or more additives. Methods for preparing such preparations can be found, for example, in Remington's Pharmaceutical Sciences (Remington: The Science and Practice of Pharmacy, 19th ed., A.R. Gennaro (ed), Mack Publishing Co., N.J., 1995; R.
  • the nucleic acid molecules e.g., mRNA
  • the nucleic acid molecules can be delivered by physical, biological, or chemical methods (see, e.g., S. Guan, J. Rosenecker, Gene Ther. 2017, 24, 133.).
  • Physical methods include, but are not limited to, delivery by gene gun (e.g., with Au-particles), electroporation, sonoporation, etc. (see, e.g., Kutzler et al., (2008) DNA vaccines: Ready for prime time? Nat Rev Genet 9:776–788; Geall et al., Nonviral delivery of self-amplifying RNA vaccines. Proc Natl Acad Sci US A. Sep. 4, 2012; 109(36):14604-9.).
  • Biological methods include, but are not limited to, delivery via viral vectors (eg, retroviral vectors, adenoviral vectors, adeno-associated viral vectors).
  • viral vectors eg, retroviral vectors, adenoviral vectors, adeno-associated viral vectors.
  • Chemical methods include, but are not limited to, delivery via natural proteins/glycans, polymers, and lipids.
  • natural proteins/glycans include protamine and chitosan (see, e.g., AE et al., Cancer Immunol. Immunother. 2015, 64, 1461; US Kumar et al. ACS Nano 2021, 11, 17582).
  • Exemplary polymers include polyethyleneimine (PEI) (e.g., linear PEI, branched PEI, and dendritic PEI), poly(beta-aminoester) (PBAE) (see, e.g., K. Singha et al., Nucleic Acid Ther. 2011, 21, 133; AA Eltoukhy et al., Biomaterials 2012, 33, 3594).
  • PEI polyethyleneimine
  • PBAE poly(beta-aminoester)
  • Exemplary lipids include cationic lipids such as 1,2-diocta-decenyl-3-trimethylammonium-propane (DOTMA) and 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) (see, e.g., X. Hou et al. al., Nat. Rev. Mater. 2021, 10, 1078.), liposomes formed from DOTMA, DOTAP and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, auxiliary lipid), which can Self-assembled with mRNA to form colloidally stable nanoparticles (see, for example, L.M. Kranz et al., Nature 2016, 534, 396.).
  • DOTMA 1,2-diocta-decenyl-3-trimethylammonium-propane
  • DOTAP 1,2-dioleoyl-3-trimethylammoniumpropane
  • 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, e.g., S.C. Sample et al., Nat. Biotechnol. 2010, 28, 172). After cellular internalization, nanoparticles formed from ionizable lipids are encapsulated in endosomes.
  • ionizable lipids obtain protons for ionization, thus promoting the fusion of lipid nanoparticles (LNPs) with the endosomal membrane, ultimately allowing the lipid nanoparticles (LNPs) to be loaded onto the endosomal membrane.
  • LNPs lipid nanoparticles
  • the mRNA on the plasma nanoparticles is released into the cytoplasm (see, for example, L. Miao et al., Mol. Cancer 2021, 20, 41.).
  • Ionizable lipids can be combined with cholesterol, accessory lipids, and pegylated lipids (ie, PEGylated lipids) to form lipid nanoparticle formulations.
  • Cholesterol is a naturally rigid and hydrophobic lipid that maintains the structure and stability of lipid nanoparticles. It can also promote the fusion of mRNA-loaded lipid nanoparticles (i.e., mRNA nanoparticles) with endosomal membranes.
  • Auxiliary lipids such as the zwitterionic lipids DOPE, 1,2-distearoyl-snglycero-3-phosphocholine (DSPC) and 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) are widely used to facilitate cell membrane penetration and endosomal membrane escape (see, for example, N. Chaudhary et al., Nat. Rev. Drug Discovery 2021, 20, 817.).
  • PEGylated lipids are composed of PEG and anchoring lipids. Hydrophilic PEG is mainly distributed in mRNA The surface of the complex, while the hydrophobic region is embedded inside the lipid bilayer.
  • the introduction of PEGylated lipids not only increases the half-life of lipid nanoparticles but also modulates the particle size by changing the molecular weight of the PEG chain.
  • the molecular weight and lipid tail length can range from 350 to 3000 Da and 10 to 18 carbons respectively (see, e.g., N. Chaudhary et al., Nat. Rev. Drug Discovery 2021, 20, 817.).
  • DLin-MC3-DMA is a key component of Onpattro approved by the U.S. Food and Drug Administration (FDA) for siRNA delivery
  • FDA U.S. Food and Drug Administration
  • DLin-MC3-DMA has also been widely used for mRNA delivery, including protein and peptide replacement, gene editing, and antiviral infection (see, e.g., RS Riley et al., Sci. Adv. 2021, 7, eaba1028.).
  • SM-102 and ALC-0315 two “star molecules”, have been approved by the FDA as key ingredients in the BNT162b and mRNA-1273 vaccines, respectively, to prevent COVID-19 (see, e.g., X. Hou et al., Nat. Rev. .Mater.2021,10,1078.).
  • An ideal lipid-based mRNA carrier must meet the following conditions: 1) Naked mRNA can form a stable complex to protect the mRNA from degradation; 2) Four key components (ionizable lipids, cholesterol, auxiliary lipids) should be added (protonated and PEGylated lipids) to stabilize the mRNA complex; 3) components of the lipid nanoparticle should be protonated to induce membrane destabilization and promote endosomal escape of the mRNA complex; and 4) all lipid materials They are all biodegradable and will not cause any harm to the patient.
  • lipid-based delivery platforms 1) Degradability of ionizable lipids: The backbone structure of lipids promotes lipid clearance and promotes lipid clearance by introducing alkynyl and ester groups into the lipid tail. Reduce toxicity; 2) Immunogenicity of lipid nanoparticles: Heterocyclic lipids within lipid nanoparticles can improve the efficiency of mRNA vaccines by activating the stimulator of interferon genes (STING) pathway in dendritic cells (DC) (See, for example, L. Miao et al., Nat. Biotechnol. 2019, 37, 1174.); 3) Stability of lipid nanoparticles: Some promising strategies are expected to improve the stability of mRNA vaccines, including pK a Optimization, excipient introduction and mRNA modification, etc.
  • STING interferon genes
  • Lipid nanoparticles can be produced using components, compositions and methods as are well known in the art, see for example PCT/US2016/052352, PCT/US2016/068300, PCT/US2017/037551, PCT/US2015/027400, PCT/US2016/ 047406, PCT/US2016/000129, PCT/US2016/014280, PCT/US2016/014280, PCT/US2017/038426, PCT/US2014/027077, PCT/US2014/055394, PCT/US2016/052117, PCT /US2012/069610 ⁇ PCT/US2017/027492, CT/US2016/059575 and PCT/US2016/069491, the entire contents of which are incorporated herein by reference.
  • the present application provides an oncolytic virus capable of expressing a chimeric protein construct described herein.
  • oncolytic virus refers to any virus that is capable of infecting tumor cells, replicating in the tumor cells, and lysing the tumor cells. In certain embodiments, the oncolytic virus is further capable of spreading to other tumor cells in successive replication cycles.
  • Oncolytic viruses can be derived from a variety of viruses, non-limiting examples of which include vaccinia virus, adenovirus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), myxoma virus, reovirus, poliovirus viruses, vesicular stomatitis virus (VSV), measles virus (MV), Lassa virus (LASV) and Newcastle disease virus (NDV), and variants of these viruses.
  • viruses non-limiting examples of which include vaccinia virus, adenovirus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), myxoma virus, reovirus, poliovirus viruses, vesicular stomatitis virus (VSV), measles virus (MV), Lassa virus (LASV) and Newcastle disease virus (NDV), and variants of these viruses.
  • viruses non-limiting examples of which include vaccinia virus, adeno
  • the application provides an oncolytic virus comprising a nucleic acid molecule as provided herein operably linked to at least one polynucleotide regulatory element (e.g., a promoter) to express the The chimeric protein construct encoded by the nucleic acid molecule.
  • a polynucleotide regulatory element e.g., a promoter
  • the oncolytic viruses described herein can infect essentially any cell type.
  • oncolytic viruses can be replication selective. Replication-selective oncolytic viruses replicate more in tumor cells than in non-tumor cells.
  • oncolytic viruses can be made replication selective if their replication is under the control of gene expression regulators, such as the enhancer/promoter region derived from the 5' side of the albumin gene (see e.g. Miyatake et al. Human, 1997, J. Virol. 71: 5124-5132).
  • gene expression regulators such as the enhancer/promoter region derived from the 5' side of the albumin gene (see e.g. Miyatake et al. Human, 1997, J. Virol. 71: 5124-5132).
  • an oncolytic virus may have a tumor cell-specific promoter, or tumor cell-specific transcriptional regulatory sequences in its genome.
  • Tuor cell specific with respect to a promoter or transcriptional sequence means that it is typically present in target tumor cells at higher levels than in normal cells. In this way, oncolytic viruses can be conferred with enhanced levels of tumor cell specificity.
  • the major transcription unit of HSV can be placed under the transcriptional control of the tumor growth factor- ⁇ (TGF- ⁇ ) promoter.
  • TGF- ⁇ tumor growth factor- ⁇
  • Certain tumor cells are known to overexpress TGF- ⁇ relative to non-tumor cells of the same type. Therefore, an oncolytic virus in which replication is transcriptionally controlled by the TGF- ⁇ promoter is replication-selective in that it replicates better in certain tumor cells than in non-tumor cells of the same type. Similar replication-selective oncolytic viruses can be prepared using any gene expression modulator known to cause overexpression selectively in affected cells.
  • Replication-selective oncolytic viruses can be, for example, HSV-1 mutants in which the gene encoding ICP34.5 is mutated or deleted.
  • Oncolytic viruses can further contain other modifications in their genomes.
  • oncolytic viruses can contain additional DNA inserted into the UL44 gene. This insertion can produce functional inactivation of the UL44 gene and the resulting lytic phenotype, or it can insert into an already inactive gene or replace a missing gene.
  • the oncolytic virus is engineered to place the nucleic acid sequence encoding the chimeric protein construct described herein under the control of a tumor cell-specific promoter.
  • the oncolytic virus is engineered to place a gene encoding at least one protein required for viral replication under the control of a tumor cell-specific promoter.
  • the oncolytic viral vector can express a chimeric protein construct described herein whose targeting domain is capable of recognizing and binding tumor proteins.
  • the tumor protein can be a protein that is highly expressed in tumor cells (e.g., c-Myc, Bcl-2, Bcl-xL, Bcl-w, KRAS), which is expressed in tumor cells and promotes the development of tumor cells.
  • Growth proteins such as estrogen receptors, androgen receptors, Her2, VEGF, VEGFR, PDGFR ⁇ , EGFR, EGFR mutants
  • proteins that promote tumor cells to escape normal immune responses such as PD-L1, TGF- ⁇ 1 .
  • the targeting domain of the chimeric protein construct can bind Bcl-2.
  • Any targeting domain capable of binding bcl-2 can be used.
  • the ER-TPD element targeting the Bcl-2 family protein is constructed and cloned and loaded behind the strong promoter of the oncolytic virus. After specifically infecting tumor cells, the oncolytic virus amplifies in large quantities and expresses Bcl.
  • the ER-TPD element of -2 family proteins realizes the degradation of intracellular Bcl-2 anti-apoptotic member proteins, directly leading to tumor cell apoptosis.
  • the targeting domain of the chimeric protein construct can bind VEGFR.
  • Any targeting domain capable of targeting VEGFR can be used, such as, but not limited to, heavy chain and light chain recombinant scFv tandem ER- of VEGFR antibodies (anti-VEGF-A antibody Bevacizumab and anti-VEGFR2 antibody Ramucizumab,) TPD elements, construct targeting VEGFR ER-TPD elements, and clone and load them on the strong promoter of oncolytic viruses. After specifically infecting tumor cells, the oncolytic viruses amplify in large quantities and express VEGFR ER-TPD elements, directly inhibiting tumors. The expression of VEGFR in cells leads to growth inhibition and apoptosis of tumor cells due to lack of sufficient nutrients.
  • the targeting domain of the chimeric protein construct can bind TGF- ⁇ 1 and PD-L1.
  • Any targeting domain capable of targeting bcl-2 can be used, for example, but not limited to, using recombinant heavy and light chain scFv tandem ER-TPD elements of TGF- ⁇ 1 and PD-L1 antibodies to construct TGF-targeting - ⁇ 1 and PD-L1ER-TPD elements are cloned and loaded into the strong promoter of oncolytic virus. After specifically infecting tumor cells, the oncolytic virus amplifies in large quantities and expresses TGF- ⁇ 1 and PD-L1ER-TPD elements. Directly inhibit and degrade the expression of TGF- ⁇ 1 and PD-L1 in tumor cells, break the tumor immunosuppressive microenvironment, and achieve the effect of treating tumors.
  • the chimeric protein construct of the present application has unique advantages when expressed in tumor cells through oncolytic viral vectors.
  • the chimeric protein construct of the present application targets and degrades proteins in tumor cells that help tumor cells grow or immune escape or resist apoptosis, which can promote the apoptosis or death of tumor cells;
  • oncolytic virus vectors It can specifically replicate in tumor cells, promote tumor cell lysis through viral replication and lysis, and integrate two different tumor killing methods into an oncolytic virus vector to achieve a synergistic killing effect in the same drug form.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the oncolytic virus provided by the present disclosure and a pharmaceutically acceptable vehicle.
  • pharmaceutically acceptable vehicles include any and all transportation media, solvents, diluents, excipients, adjuvants, dispersion media, coatings, antibacterial and antifungal agents, absorbents, etc. Such substances are compatible for administration to animals and especially human subjects.
  • the oncolytic viruses described herein, or pharmaceutical compositions thereof are formulated for intravenous or intratumoral administration.
  • oncolytic viruses or pharmaceutical compositions thereof can be placed in a solvent or diluent suitable for human or animal use.
  • the solvent or diluent is preferably isotonic, hypotonic or slightly hypotonic and has a relatively low ionic strength.
  • Typical examples include sterile water, physiological saline (such as sodium chloride), Ringer’s solution, glucose, trehalose or sucrose solutions, Hank’s solution and other aqueous physiological balanced salt solutions.
  • the oncolytic virus or pharmaceutical composition thereof described herein may have a buffering agent.
  • Suitable buffers include, but are not limited to, phosphate buffer (eg, PBS), bicarbonate buffer, and/or Tris buffer capable of maintaining a physiological or slightly alkaline pH (eg, from about pH 7 to about pH 9).
  • the oncolytic virus composition of the present invention may be frozen (eg -70°C, -20°C) to improve its stability, especially under production conditions and long-term (ie at least 6 months, preferably at least 2 years) Prepared for stability during storage, refrigerated storage (e.g. 4°C), and room temperature storage.
  • frozen liquid form e.g WO98/02522 and WO2008/114021, etc.
  • Solid (eg dry powder or lyophilized) compositions can be obtained by steps involving vacuum drying and lyophilization.
  • buffer preparations with the addition of NaCl and sugar are particularly suitable for preserving viruses.
  • the present disclosure also provides a cellular pharmaceutical composition, which includes the engineered cells or cell populations provided by the present disclosure and a pharmaceutically acceptable medium.
  • exemplary vehicles include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants ; chelating agents, such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); and preservatives.
  • the cellular pharmaceutical compositions of the invention are formulated for intravenous administration.
  • the present disclosure also provides a use of the chimeric protein construct, nucleic acid molecule or vector described herein in preparing cells to be transplanted (eg, allogeneic cell transplantation).
  • the cells to be transplanted are immune cells (eg, T cells), stem cells (and differentiated cells thereof), kidney cells, islet cells, cardiomyocytes, etc.
  • the cells to be transplanted may be autologous cells or allogeneic cells.
  • the present application also provides a cell to be transplanted that expresses the chimeric protein construct, nucleic acid molecule or vector described above, wherein the chimeric protein construct described in the present application is expressed, and the chimeric protein
  • the construct includes an ERAD mechanism protein-binding domain and a targeting domain that includes a domain that specifically targets or binds a target protein, which is a transplant rejection-related protein.
  • the chimeric protein construct further comprises a protein degradation pathway member (eg, E3 ubiquitin ligase, proteasome, lysosome) binding domain as described herein. Any protein degradation pathway member binding domain described herein may be used.
  • the transplant rejection-related proteins include antigen presenting molecules (such as MHC class I molecules, MHC class II molecules, MICA/B molecules, etc.), antigen recognition molecules (such as TCR, CD123, NKG2D, etc.), Immune checkpoint molecules (such as PD-1, PD-L1, CTLA4, TIM3, TIGIT, LAG3, A2AR, BTLA, IDO1, IDO2, TDO, KIR, NOX2, VISTA, SIGLEC7, PVR, etc.), etc.
  • antigen presenting molecules such as MHC class I molecules, MHC class II molecules, MICA/B molecules, etc.
  • antigen recognition molecules such as TCR, CD123, NKG2D, etc.
  • Immune checkpoint molecules such as PD-1, PD-L1, CTLA4, TIM3, TIGIT, LAG3, A2AR, BTLA, IDO1, IDO2, TDO, KIR, NOX2, VISTA, SIGLEC7, PVR, etc.
  • the transplant rejection-associated protein includes an immunogenic protein (eg, HLA (HLA ⁇ / ⁇ ), TCR ( ⁇ TCR), NKG2D (natural killer cell family 2 member D) ligand, etc.).
  • an immunogenic protein eg, HLA (HLA ⁇ / ⁇ ), TCR ( ⁇ TCR), NKG2D (natural killer cell family 2 member D) ligand, etc.
  • the present disclosure also provides the above-mentioned cells to be transplanted and their treatment methods, which can overcome the problems faced in existing cell transplantation and cell therapy such as the source of personalized transplant cells and treatment cells, inability to standardize and scale production, and low treatment efficiency.
  • the immune system has great plasticity and a nearly unlimited ability to detect invading viruses, bacteria, foreign cells, and diseased cells.
  • This extraordinary immune monitoring ability is mainly achieved through humoral immunity and cellular immunity, which includes two important molecular structures: immunoglobulin and T cell receptor (TCR).
  • TCR is the defining structure of T cells and is a transmembrane heterodimer composed of ⁇ and ⁇ chains or ⁇ and ⁇ chains linked by disulfide bonds. Within these chains, the complementarity determining regions (CDRs) determine the antigen to which the TCR will bind.
  • CDRs complementarity determining regions
  • TCR ⁇ and TCR ⁇ subunits or TCR ⁇ and TCR ⁇ in ⁇ T cells
  • MHC major histocompatibility complex
  • the human MHC class I gene region includes alleles of HLA-A, B, and C loci, encoding classic class I antigens (molecules) such as HLA-A antigen, B antigen, and C antigen, called HLA-A and HLA-B. and HLA-C.
  • HLA-A antigen, B antigen, and C antigen called HLA-A and HLA-B. and HLA-C.
  • These antigen molecules are present on the surface of all body cells and bind to intracellular protein epitope peptides for recognition by the immune system. If cells produce mutant proteins, or are invaded by foreign bacteria or viruses, after the cells present these mutant proteins or heterologous protein epitopes, immune cells will recognize them and carry out immune attack and killing, thereby eliminating diseased cells, bacteria and virus invasion. cell.
  • ⁇ / ⁇ T lymphocytes recognize peptide-MHC ligands through a collection of polyproteins called the ⁇ T cell antigen receptor (TCR) CD3 complex.
  • TCR ⁇ T cell antigen receptor
  • the structure consists of a variable ⁇ TCR dimer that binds the antigen and three invariant dimers (CD3 ⁇ , ⁇ , and ⁇ ) involved in TCR.CD3 surface transport, stabilization, and signal transduction.
  • the ⁇ T cell receptor ( ⁇ TCR) is expressed on the majority (approximately 95%) of T cells and plays a key role in T cell activation by recognizing major histocompatibility complex (MHC) anchored antigens.
  • MHC major histocompatibility complex
  • TCR-mediated T cell activation is a key step in the pathogenesis of graft-versus-host disease (GVHD) during allogeneic hematopoietic cell transplantation (allo-HCT) and allogeneic CAR-T cell therapy.
  • GVHD graft-versus-host disease
  • allo-HCT allogeneic hematopoietic cell transplantation
  • allogeneic CAR-T cell therapy allogeneic CAR-T cell therapy.
  • HLA human leukocyte antigen
  • MHC human major histocompatibility complex
  • HLA mismatch occurs when the donor HLA on the allograft is different from the recipient. HLA mismatch results in activation of alloreactive T cells, which can cause acute cellular rejection (ACR) within six months of transplantation.
  • mismatched donor HLA antigens are also targets for the development of de novo donor-specific HLA antibodies (dnDSA), which play an enhancing role in acute and chronic transplant cell (e.g., T cell) rejection.
  • dnDSA de novo donor-specific HLA antibodies
  • cells to be transplanted expressing the chimeric protein constructs, nucleic acid molecules or vectors described above have reduced immunogenicity.
  • the cells to be transplanted with reduced immunogenicity (or high compatibility) described in the present application are T cells, and the TPD chimeric protein constructs expressed by them include specific recognition TCR and/or HLA The scFv as well as the transmembrane domain or functional variants thereof and the endoplasmic reticulum resident domain or functional variants thereof of the HCMV glycoproteins US2 and/or US11.
  • the ERAD mechanism protein binding domain comprises the amino acid sequence shown in SEQ ID NO. 112 or SEQ ID NO. 118 or is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) thereof. ) identity of the amino acid sequence.
  • the cells to be transplanted with reduced immunogenicity are T cells expressing an ER-TPD chimeric protein construct comprising an scFv that specifically recognizes TCR and a transmembrane structure of adenovirus E3-K19 domain or functional variant thereof and endoplasmic reticulum resident domain or functional variant thereof.
  • the cells to be transplanted with reduced immunogenicity are T cells expressing an ER-TPD chimeric protein construct comprising a targeting protein binding domain and an ERAD machinery protein binding domain
  • said Targeting protein binding domains include those contained in 6 CDRs of an amino acid sequence as set forth in SEQ ID NO: 152 or an amino acid sequence having at least 80% (eg, 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. 96 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 above-described T cell expressed ER-TPD chimeric protein construct with reduced immunogenicity further comprises a protein degradation pathway member (e.g., E3 ubiquitin ligase, proteasome) binding domain, optionally , the protein degradation pathway member binding domain is connected to the ERAD mechanism protein binding domain.
  • the above-described T cell expressed ER-TPD chimeric protein construct with reduced immunogenicity further comprises an E3 ubiquitin ligase binding domain having the amino acid sequence set forth in SEQ ID NO: 162.
  • the above-described T cell expressed ER-TPD chimeric protein construct with reduced immunogenicity further comprises an E3 ubiquitin ligase binding domain having the amino acid sequence set forth in SEQ ID NO: 166.
  • the cells to be transplanted eg, T cells
  • the cells to be transplanted also express a co-expression moiety as described above.
  • proteins capable of degrading or reducing MHC class I or class II molecules can be expressed as co-expression moieties in the cells to be transplanted (such as T cells) with reduced immunogenicity.
  • T cells such as T cells
  • intact viral ER-resident glycoproteins including, but not limited to, HCMV US2, US3, US11, US10, adenovirus E3-K19, HCMV US6, and HSV ICP47.
  • Effective down-regulation of TCR will greatly inhibit TCR-mediated immune attack and reduce GVHD during allogeneic infusion of T cells.
  • Including native viral ER-resident glycoproteins can further inhibit MHC molecules, thereby preventing peptide presentation to recipient CD8+ T cells and inhibiting immune recognition of allogeneic T cells. Therefore, the highly compatible T cells as mentioned above are also called “autologous universal T cells (UT)” or “universal T cells (UT)", which can achieve the purpose of allogeneic blood transfusion therapy, and It can improve the compatibility and long-term persistence of allogeneic T cells after infusion.
  • UT autologous universal T cells
  • UT universal T cells
  • the present disclosure provides a method of reducing a subject's immune response to cell transplantation, comprising administering to the subject a therapeutically effective amount of cells to be transplanted with reduced immunogenicity as described above, thereby The subject's immune response to the graft or to the modified cells is reduced.
  • the cells to be transplanted intracellularly express a TPD chimeric protein construct as described herein, the TPD chimeric protein construct comprising a targeted immunogenic protein (e.g., HLA (HLA ⁇ / ⁇ ), TCR ( ⁇ TCR), NKG2D (natural killer cell family 2 member D) ligand, etc.) and the ERAD mechanism protein binding domain described above, and optionally, the protein degradation pathway member binding domain .
  • a targeted immunogenic protein e.g., HLA (HLA ⁇ / ⁇ ), TCR ( ⁇ TCR), NKG2D (natural killer cell family 2 member D) ligand, etc.
  • Cell therapy uses genetic engineering technology to modify cells (such as immune cells) so that they can present tumor-related antigens or express receptors that specifically recognize disease cells. They are exponentially amplified in vitro and then reinfused into the patient's body to activate the body's immune system to respond to the disease. The efficacy of attacking tumor cells or directly specifically recognizing and killing disease cells.
  • CD19-targeted CAR-T cells achieved the first targeted elimination of tumor cells in patients with B-cell leukemia in the history of human medicine. It became another treatment technology that can truly cure leukemia after bone marrow stem cell transplantation, and opened the door to precision medicine cell therapy. new era. This technology is expected to be applied in the treatment of various hematological tumors and solid tumors.
  • T cells are derived from the patient itself. If the patient has received a large amount of radiotherapy and chemotherapy, etc., the immune system function is impaired, and it is difficult to obtain it from the patient's periphery. A sufficient number of T cells are isolated from the blood. Even if they are isolated and modified, the proliferation and killing functions of the T cells are still very weak, so it is difficult to exert a therapeutic effect.
  • the cells to be transplanted (such as T cells) provided in this application can overcome the problems faced in existing cell therapy such as tumor target differences, tumor immunosuppressive environment, and sources of personalized treatment cells.
  • the cells to be transplanted with reduced immunogenicity can be further genetically engineered, for example to express as above
  • the co-expression part is used to achieve therapeutic purposes.
  • the cells to be transplanted can be Express binding domains or molecules that recognize or bind disease-related target proteins, such as chimeric antigen receptor CAR, engineered TCR, or CNK receptors provided in this application, or antiviral protein binding domains, etc.
  • molecules capable of mediating the migration of the disease to be transplanted to the disease microenvironment can also be expressed in the cells to be transplanted (such as T cells) with reduced immunogenicity. receptor).
  • immunostimulatory molecules can be expressed in the cells to be transplanted (such as T cells) with reduced immunogenicity.
  • this application also provides the method of introducing NK elements, especially optimized recombinant NK elements, into the cells to be transplanted (such as UT cells) provided by this application, so that T cells can recognize as efficiently and broadly as NK cells.
  • NK targets include family member proteins such as MICA, MICB, and ULBP1–6, which can be widely expressed in various types of tumor cells and cover different stages of tumor progression, CNK technology can effectively solve the off-target effects of a single CAR-T and eliminate The probability of tumor immune escape.
  • the Chimeric Adapter introduced by CNK in the design can effectively transduce and amplify NK signals, overcome the limitations of immune checkpoints such as PD1 signals, efficiently activate T cells, and achieve the target of tumor cells. Kill.
  • the Chimeric Adapter amplifies CNK-T cell signals, resists immunosuppression in the tumor environment, and achieves T cell Activation to kill tumor cells; CNK-T cells recognize targets through NK and can also eliminate immunosuppressive cells such as MDSC.
  • the virus infects cells it expresses specific functional proteins, and the assembly or transport of MHCI may directly promote the directional degradation of MHCI molecules, thereby inhibiting the presentation of viral antigenic epitopes and causing immune evasion.
  • the present disclosure provides a UT cell that also expresses a CNK receptor and optionally a CAR, which may also be referred to as a "CNKT-UT.”
  • CNK-UT has broad-spectrum tumor recognition and killing capabilities.
  • This application further designs a compound specific target CAR/CNK-UT product, demonstrating that the CAR/CNK-UT product has more powerful killing and activating functions for tumor cells than conventional CAR-T; in animal experiments, the CAR/CNK-UT product It also has more efficient tumor removal capabilities.
  • UT technology realizes allogeneic and universal transformation, and T cells are derived from healthy donors, thus realizing the standardization and large-scale production of CNK-UT products, which can be prepared in advance and ensure the function of T cells to kill and activate tumor cells.
  • 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. 158, preferably 85%, 90 %, 95%, 96%, 97%, 98%, 99% or above identical amino acid sequences.
  • the CNKT-UT comprises a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 158.
  • the present disclosure also provides the use of CNKT-UT in the preparation of a medicament for treating a disease.
  • the disclosure also provides an immune cell comprising or expressing a chimeric protein construct, a nucleic acid molecule or a vector encoding the same described herein, wherein the chimeric protein construct comprises an ERAD mechanism protein binding domain and a targeting domain comprising a domain that specifically targets or binds a target protein expressed on the immune cell as well as on the target cell.
  • the immune cell further comprises or expresses a binding domain or molecule (eg, CAR), a nucleic acid molecule encoding the same, or a vector that targets the target protein.
  • examples of such target proteins include CD123, CD5, CD7, CD38, or CD4.
  • target proteins are expressed both on target cells (eg, cancer cells or virus-infected cells) and on immune cells used for therapy. Such targets are usually difficult to treat through cell therapy. Therefore, when the immune cells used for treatment express a CAR targeting such targets, the CAR will also cause recognition of the same target expressed by the immune cells themselves, leading to immune Cells undergo autophagy and cannot expand normally, or have limited killing effect on target cells.
  • target proteins expressed by immune cells themselves can be degraded, thereby avoiding autophagy while retaining the specific killing effect on target cells.
  • Another advantage of using the chimeric protein construct of the present application is that the chimeric protein construct targeting the target and the binding domain (such as CAR, TCR, etc.) targeting the target can be expressed simultaneously through co-expression. ), which is simpler and more effective than traditional gene knockout and other methods.
  • the chimeric protein construct further comprises a protein degradation pathway member (eg, E3 ubiquitin ligase, proteasome, lysosome) binding domain as described herein. Any protein degradation pathway member binding domain described herein may be used.
  • a protein degradation pathway member eg, E3 ubiquitin ligase, proteasome, lysosome
  • Cell administration methods for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions.
  • methods of adoptive T cell therapy are described 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) .
  • cells or individual populations or subtypes of cells are administered to a subject in the range of about one million to about one hundred billion cells, such as 1 million to about 50 billion cells (e.g., about 500 Ten thousand 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 any two of the above range defined by values), for example, about 10 million to about 100 billion cells (for example, about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 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, from about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells cells, about 800 million cells, about 900 million cells, about 3 billion cells
  • the dose of total cells and/or the dose of individual subpopulations of cells is between at or about 10 4 cells/kilogram (kg) body weight to at or about 10 9 cells/kilogram (kg) body weight Within the range, for example, between 10 5 and 10 6 cells/kg body weight, for example, at least or at least about or at or about 1 ⁇ 10 5 cells/kg, 1.5 ⁇ 10 5 cells/kg, 2 ⁇ 10 5 cells/kg or 1 ⁇ 10 6 cells/kg body weight.
  • the cells are administered at or within a range of between or about 10 4 and at or about 10 9 T cells/kilogram (kg) of body weight, e.g., between 10 5 and 10 6 T cells /kg body weight, such as at least or at least about or at or about 1 ⁇ 10 5 T cells/kg, 1.5 ⁇ 10 5 T cells/kg, 2 ⁇ 10 5 T cells/kg, or 1 ⁇ 10 6 T cells/kg body weight.
  • body weight e.g., between 10 5 and 10 6 T cells /kg body weight, such as at least or at least about or at or about 1 ⁇ 10 5 T cells/kg, 1.5 ⁇ 10 5 T cells/kg, 2 ⁇ 10 5 T cells/kg, or 1 ⁇ 10 6 T cells/kg body weight.
  • the cells may be administered by any suitable means, such as by bolus injection, by injection, such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, Intrachoroidal injection, intracameral injection, subperineal injection, subconjunctival injection, sub-Tenon injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery.
  • they are administered parenterally, intrapulmonary, and intranasally, and if local treatment is desired, intralesional administration.
  • Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • a given dose is administered by administering the cells as a single bolus. In some embodiments, it is administered by administering the cells as multiple bolus injections, for example, over a period of no more than 3 days, or by continuous infusion of the administered cells.
  • repeated dosing methods are provided wherein a first dose of cells is administered followed by one or more second consecutive doses.
  • multiple doses of cells are typically timed and sized to increase the efficacy and/or activity and/or function of antigen-expressing T cells (eg, CAR-expressing T cells).
  • repeated administration reduces the effects of inhibitory immune molecules such as PD-1 and/or or the down-regulation or inhibitory activity that can occur when PD-L1 is up-regulated on antigen-expressing, e.g., CAR-expressing T cells.
  • Methods include administering a first dose, usually followed by one or more consecutive doses, with a specified time frame between doses.
  • administration of a given "dose” includes administration of a given amount or number of cells as a single composition and/or as a single uninterrupted administration (e.g., as a single injection or continuous infusion), and also includes the administration of a given amount or number of cells as divided doses in multiple individual compositions or infusions over a specified period of time (not to exceed 3 days).
  • the first or sequential dose is a single or sequential administration of a specified number of cells administered or initiated at a single time point.
  • the first or subsequent doses are administered as multiple injections or infusions over a period of not more than three days, such as three or two days, once daily, or as multiple infusions over a single day. .
  • Cell therapy uses genetic engineering technology to modify cells (such as immune cells) so that they can present tumor-related antigens or express receptors that specifically recognize disease cells. They are exponentially amplified in vitro and then reinfused into the patient's body to activate the body's immune system to respond to the disease. The efficacy of attacking tumor cells or directly specifically recognizing and killing disease cells.
  • CD19-targeted CAR-T cells achieved the first targeted elimination of tumor cells in patients with B-cell leukemia in the history of human medicine. It became another treatment technology that can truly cure leukemia after bone marrow stem cell transplantation, and opened the door to precision medicine cell therapy. new era. This technology is expected to be applied in the treatment of various hematological tumors and solid tumors.
  • T cells are derived from the patient itself. If the patient has received a large amount of radiotherapy and chemotherapy, etc., the immune system function is impaired, and it is difficult to obtain it from the patient's periphery. A sufficient number of T cells are isolated from the blood. Even if they are isolated and modified, the proliferation and killing functions of the T cells are still very weak, so it is difficult to exert a therapeutic effect.
  • the cells to be transplanted (such as T cells) provided in this application can overcome the problems faced in existing cell therapy such as tumor target differences, tumor immunosuppressive environment, and sources of personalized treatment cells.
  • the cells to be transplanted with reduced immunogenicity can be further genetically engineered, for example to express as above
  • the co-expression part is used to achieve therapeutic purposes.
  • the cells to be transplanted can be Express binding domains or molecules that recognize or bind disease-related target proteins, such as chimeric antigen receptor CAR, engineered TCR, or CNK receptors provided in this application, or antiviral protein binding domains, etc.
  • molecules capable of mediating the migration of the disease to be transplanted to the disease microenvironment can also be expressed in the cells to be transplanted (such as T cells) with reduced immunogenicity. receptor).
  • immunostimulatory molecules can be expressed in the cells to be transplanted (such as T cells) with reduced immunogenicity.
  • this application also provides the method of introducing NK elements, especially optimized recombinant NK elements, into the cells to be transplanted (such as UT cells) provided by this application, so that T cells can recognize as efficiently and broadly as NK cells.
  • NK targets include family member proteins such as MICA, MICB, and ULBP1–6, which can be widely expressed in various types of tumor cells and cover different stages of tumor progression, CNK technology can effectively solve the off-target effects of a single CAR-T and eliminate The probability of tumor immune escape.
  • the Chimeric Adapter introduced by CNK in the design can effectively transduce and amplify NK signals, overcome the limitations of immune checkpoints such as PD1 signals, efficiently activate T cells, and achieve the target of tumor cells. Kill.
  • the Chimeric Adapter amplifies CNK-T cell signals, resists immunosuppression in the tumor environment, activates T cells, and kills tumor cells; in addition, CNK-T cells recognize targets through NK It can also eliminate immunosuppressive cells such as MDSC. After the virus infects cells, it expresses specific functional proteins, and the assembly or transport of MHCI may directly promote the directional degradation of MHCI molecules, thereby inhibiting the presentation of viral antigenic epitopes and causing immune evasion.
  • the present disclosure provides a UT cell that also expresses a CNK receptor and optionally a CAR, which may also be referred to as a "CNKT-UT.”
  • CNK-UT has broad-spectrum tumor recognition and killing capabilities.
  • This application further designs a compound specific target CAR/CNK-UT product, demonstrating that the CAR/CNK-UT product has more powerful killing and activating functions for tumor cells than conventional CAR-T; in animal experiments, the CAR/CNK-UT product It also has more efficient tumor removal capabilities.
  • UT technology realizes allogeneic and universal transformation, and T cells are derived from healthy donors, thus realizing the standardization and large-scale production of CNK-UT products, which can be prepared in advance and ensure the function of T cells to kill and activate tumor cells.
  • 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. 158, preferably 85%, 90 %, 95%, 96%, 97%, 98%, 99% or above identical amino acid sequences.
  • the CNKT-UT comprises a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 158.
  • the present disclosure also provides the use of CNKT-UT in the preparation of a medicament for treating a disease.
  • the disclosure also provides an immune cell comprising or expressing a chimeric protein construct, a nucleic acid molecule or a vector encoding the same described herein, wherein the chimeric protein construct comprises an ERAD mechanism protein binding domain and a targeting domain comprising a domain that specifically targets or binds a target protein expressed on the immune cell as well as on the target cell.
  • the immune cell further comprises or expresses a binding domain or molecule (eg, CAR), a nucleic acid molecule encoding the same, or a vector that targets the target protein.
  • examples of such target proteins include CD123, CD5, CD7, CD38, or CD4.
  • target proteins are expressed both on target cells (eg, cancer cells or virus-infected cells) and on immune cells used for therapy. Such targets are usually difficult to treat through cell therapy. Therefore, when the immune cells used for treatment express a CAR targeting such targets, the CAR will also cause recognition of the same target expressed by the immune cells themselves, leading to immune Cells undergo autophagy and cannot expand normally, or have limited killing effect on target cells.
  • target proteins expressed by immune cells themselves can be degraded, thereby avoiding autophagy while retaining the specific killing effect on target cells.
  • Another advantage of using the chimeric protein construct of the present application is that the chimeric protein construct targeting the target and the binding domain (such as CAR, TCR, etc.) targeting the target can be expressed simultaneously through co-expression. ), which is simpler and more effective than traditional gene knockout and other methods.
  • the chimeric protein construct further comprises a protein degradation pathway member (eg, E3 ubiquitin ligase, proteasome, lysosome) binding domain as described herein. Any protein degradation pathway member binding domain described herein may be used.
  • a protein degradation pathway member eg, E3 ubiquitin ligase, proteasome, lysosome
  • Cell administration methods for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions.
  • methods of adoptive T cell therapy are described 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) .
  • cells or individual populations or subtypes of cells are administered to a subject in the range of about one million to about one hundred billion cells, such as 1 million to about 50 billion cells (e.g., about 500 Ten thousand 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 any two of the above range defined by values), for example, about 10 million to about 100 billion cells (for example, about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 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, from about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells cells, about 800 million cells, about 900 million cells, about 3 billion cells
  • the dose of total cells and/or the dose of individual subpopulations of cells is between at or about 10 4 cells/kilogram (kg) body weight to at or about 10 9 cells/kilogram (kg) body weight Within the range, for example, between 10 5 and 10 6 cells/kg body weight, for example, at least or at least about or at or about 1 ⁇ 10 5 cells/kg, 1.5 ⁇ 10 5 cells/kg, 2 ⁇ 10 5 cells/kg or 1 ⁇ 10 6 cells/kg body weight.
  • the cells are administered at or within a range of between or about 10 4 and at or about 10 9 T cells/kilogram (kg) of body weight, e.g., between 10 5 and 10 6 T cells /kg body weight, such as at least or at least about or at or about 1 ⁇ 10 5 T cells/kg, 1.5 ⁇ 10 5 T cells/kg, 2 ⁇ 10 5 T cells/kg, or 1 ⁇ 10 6 T cells/kg body weight.
  • body weight e.g., between 10 5 and 10 6 T cells /kg body weight, such as at least or at least about or at or about 1 ⁇ 10 5 T cells/kg, 1.5 ⁇ 10 5 T cells/kg, 2 ⁇ 10 5 T cells/kg, or 1 ⁇ 10 6 T cells/kg body weight.
  • the cells may be administered by any suitable means, such as by bolus injection, by injection, such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, Intrachoroidal injection, intracameral injection, subperineal injection, subconjunctival injection, sub-Tenon injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery.
  • they are administered parenterally, intrapulmonary, and intranasally, and if local treatment is desired, intralesional administration.
  • Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • a given dose is administered by administering the cells as a single bolus. In some embodiments, it is administered by administering the cells as multiple bolus injections, for example, over a period of no more than 3 days, or by continuous infusion of the administered cells.
  • repeated dosing methods are provided wherein a first dose of cells is administered followed by one or more second consecutive doses.
  • multiple doses of cells are typically timed and sized to increase the efficacy and/or activity and/or function of antigen-expressing T cells (eg, CAR-expressing T cells).
  • repeated dosing reduces the down-regulation or inhibitory activity that can occur when inhibitory immune molecules, such as PD-1 and/or PD-L1, are up-regulated on antigen-expressing, eg, CAR-expressing, T cells.
  • Methods include administering a first dose, usually followed by one or more consecutive doses, with a specified time frame between doses.
  • administration of a given "dose” includes administration of a given amount or number of cells as a single composition and/or as a single uninterrupted administration (e.g., as a single injection or continuous infusion), and also includes the administration of a given amount or number of cells as divided doses in multiple individual compositions or infusions over a specified period of time (not to exceed 3 days).
  • the first or sequential dose is a single or sequential administration of a specified number of cells administered or initiated at a single time point.
  • the first or subsequent doses are administered as multiple injections or infusions over a period of not more than three days, such as three or two days, once daily, or as multiple infusions over a single day. .
  • the present disclosure also provides a method of degrading a target protein in vivo or in vitro, comprising delivering a nucleic acid molecule and/or vector as described herein into a cell or subject expressing the target protein, wherein Expression of the nucleic acid molecule and/or the vector in the cell or subject produces a chimeric protein construct as described herein.
  • the targeting protein binding domain of the chimeric protein construct can bind the target protein, and the ERAD mechanism protein binding domain of the chimeric protein construct can utilize the ERAD pathway to induce the ubiquitin-proteasome system (UPS) spatially. close, thereby utilizing the proteasome in the UPS to degrade the target protein.
  • UPS ubiquitin-proteasome system
  • the chimeric protein construct further includes a protein degradation pathway member binding domain, this method will induce additional degradation systems to be close to the target protein, so that the degradation effect is significantly improved.
  • the present disclosure also provides a method of treating and/or preventing recurrence of a condition or disease in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of a disorder or disease as described above.
  • Pharmaceutical compositions comprising, for example, nucleic acid molecules as described herein, such as mRNA, viral vectors, oncolytic viruses, chimeric protein constructs, etc.).
  • the method includes converting a nucleic acid molecule (e.g., mRNA) or vector encoding a chimeric protein construct described herein into (eg, a viral vector) is introduced into an individual in need thereof such that the nucleic acid molecule or vector expresses the chimeric protein construct in cells of the individual.
  • a nucleic acid molecule e.g., mRNA
  • vector encoding a chimeric protein construct described herein eg, a viral vector
  • Parenteral route is injection or infusion administration, including systemic and local routes.
  • Common types of parenteral injections are intravenous (into a vein), intraarterial (into an artery), intracutaneous (into the dermis), subcutaneous (below the epidermis), intramuscular (into a muscle), and intratumoral (intratumoral (into a muscle)). into the tumor or very close to the tumor).
  • the infusion is through the intravenous route.
  • Mucosal administration includes, but is not limited to, oral/esophageal, intranasal, tracheal, intrapulmonary, intravaginal, or intrarectal routes. Topical application may also be accomplished by transdermal means (such as patches, etc.). Conventional syringes and needles may be used for administration or any compound or device known in the art that facilitates or enhances delivery of the active agent within a subject.
  • the appropriate chimeric protein construct, its nucleic acid molecule and its vector can be selected.
  • the diseases include various types of solid tumors and hematological tumors, infectious diseases (such as viral infectious diseases), autoimmune diseases, neurodegenerative diseases, and metabolic diseases.
  • the disease is a tumor or cancer
  • the targeting domain of the chimeric protein construct includes specific targeting of a tumor-related target or an immune function-related target.
  • the tumor-related targets or immune function-related targets are as described previously in this application.
  • the solid tumor is selected from the group consisting of 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.
  • nervous system tumors include diffuse glioma, diffuse astrocytoma and anaplastic astrocytoma, glioblastoma, oligodendroglioma, oligoastrocytoma tumors, childhood diffuse gliomas, other astrocytomas, ependymomas, neuronal and mixed neuronal-glial tumors, medulloblastoma, other embryonal tumors, schwannomas, meningiomas, Solitary fibrous tumor and hemangiopericytoma, etc.
  • head and neck tumors include malignant tumors of the nasal cavity and paranasal sinuses, nasopharyngeal cancer, oral cavity cancer, laryngeal cancer, salivary gland tumors, intracranial tumors, thyroid cancer, tongue cancer, etc.
  • thoracic tumors include lung cancer, esophageal cancer, cardiac cancer, breast cancer, mediastinal tumors, and the like.
  • digestive system tumors include gastric cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, small intestinal malignant tumors, and the like.
  • genitourinary tumors include kidney cancer, prostate cancer, bladder cancer, testicular malignancy, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, and the like.
  • soft tissue and skin tumors include malignant fibrous histiocytoma, rhabdomyosarcoma, synovial sarcoma, cutaneous malignant melanoma, and the like.
  • bone tumors include osteosarcoma, Ewing's sarcoma, and the like.
  • the colon cancer is a colon adenoma.
  • the breast cancer is triple negative breast cancer cells.
  • the liver cancer is hepatocellular carcinoma.
  • the disease is a hematological neoplasm selected from the group consisting of leukemia, lymphoma (HL), multiple myeloma (MM), myelodysplastic syndrome (MDS), etc.
  • HL lymphoma
  • MM multiple myeloma
  • MDS myelodysplastic syndrome
  • the leukemia is B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute myeloid leukemia, etc.
  • the present disclosure also provides a method of treating a tumor or cancer in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of an oncolytic virus or oncolytic virus described herein.
  • Viral pharmaceutical compositions The oncolytic viruses or compositions of the invention can be administered to a subject in a single dose or in multiple doses. If multiple doses are administered, they may be administered by the same or different routes and may be administered at the same site or at different sites. It can also be administered in a continuous cycle, repeated after a rest period. The interval between each administration can range from a few hours to a year (eg, 24h, 48h, 72h, weekly, biweekly, monthly or yearly). Intervals may also be irregular (for example, after tumor development). The dosage per administration may vary within the above range.
  • the route of administration of the oncolytic virus may include intravenous and intratumoral routes.
  • the oncolytic virus may be administered once or multiple times at a suitable dose (eg, 10 7 to 5x10 9 pfu).
  • a suitable dose eg, 10 7 to 5x10 9 pfu.
  • the interval between each viral administration can range from about 1 day to about 8 weeks.
  • Another preferred treatment regimen involves 2 to 5 (eg 3) intravenous or intratumoral administrations of 10 or 10 Pfu of oncolytic vaccinia virus, approximately 1 or 2 weeks apart, which can be selected by the physician based on the actual situation.
  • the method includes administering to the subject an oncolytic viral vector comprising a nucleic acid molecule as described above, wherein the subject has cancer.
  • the pharmaceutical composition or the oncolytic virus vector containing the nucleic acid molecule as described above can degrade pathogenic proteins in a targeted manner, such as oncogenic proteins, viral proteins, immune-related proteins, etc.
  • the disease is an infectious disease
  • the targeting domain of the chimeric protein construct includes a specific targeting target associated with the infectious disease.
  • the targets related to infectious diseases are as described previously in this application.
  • viral infectious diseases include: respiratory viral diseases, gastrointestinal viral diseases, liver viral diseases, skin and mucosal viral diseases, eye viral diseases, central nervous system viral diseases, lymphatic Cellular viral diseases, insect-borne viral diseases, lentiviral infectious diseases, etc.
  • respiratory viral diseases include infections with rhinovirus, adenovirus, respiratory syncytial virus, parainfluenza virus, coronavirus, and the like; influenza; mumps, and the like.
  • gastrointestinal viral diseases include poliomyelitis; Cooksackie virus infection; ECHO virus infection; viral gastroenteritis: including rotavirus gastroenteritis, norovirus Gastroenteritis, adenovirus gastroenteritis, astrovirus gastroenteritis, coronavirus gastroenteritis and calicivirus gastroenteritis, etc.
  • the viral diseases of the liver include hepatitis A, hepatitis B, hepatitis C, hepatitis delta, hepatitis E, Epstein-Barr virus, and cytomegalovirus Viral hepatitis, etc.
  • viral diseases of the skin and mucosal membranes include measles, rubella, exanthema, varicella and shingles, smallpox, herpes simplex virus infection, rabies, foot and mouth disease, and the like.
  • ocular viral diseases include epidemic keratoconjunctivitis, follicular conjunctivitis, herpetic keratoconjunctivitis, and the like.
  • the central nervous system viral disease includes Japanese encephalitis, Western equine encephalitis, Eastern equine encephalitis, St. Louis encephalitis, Venezuelan equine encephalitis, Murray Valley encephalitis, California encephalitis inflammation, forest encephalitis and lymphocytic choriomeningitis.
  • lymphocytic viral diseases include infectious mononucleosis, cytomegalovirus infection, acquired immunodeficiency syndrome, and the like.
  • the insect-borne viral diseases include viral hemorrhagic fevers: including epidemic hemorrhagic fever, yellow fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, Argentine hemorrhagic fever, Venezuelan hemorrhagic fever, Lassa fever, Omsk hemorrhagic fever, Marburg disease and Ebola hemorrhagic fever, etc.; Dengue fever and dengue hemorrhagic fever; West Nile fever; Colorado tick-borne fever; sand fly fever, etc.
  • lentiviral infectious diseases include subacute sclerosing panencephalitis, kuru disease, progressive multifocal leukoencephalopathy, subacute spongiform encephalopathy (corticostriatal spinal cord degeneration), and the like.
  • the disease is an autoimmune disease and the targeting domain of the chimeric protein construct comprises a target associated with specific targeting of an autoantigen.
  • the autoantigen-related target points are as described previously in this application.
  • autoimmune diseases include organ-specific autoimmune diseases and systemic autoimmune diseases
  • organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, vulgaris Pemphigus, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, etc.
  • systemic autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune Hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, etc.
  • the disease is a neurological or degenerative disease
  • the targeting domain of the chimeric protein construct includes specific targeting of a target associated with the neurological disease.
  • the targets related to neurological diseases are as described previously in this application.
  • the neurological disease includes neurological disease, peripheral nerve disease such as trigeminal neuralgia, facial paralysis, hemifacial spasm, vestibular neuronitis, glossopharyngeal neuralgia, mononeuropathy, brachial plexus neuralgia, polyneuropathy Mononeuropathy, polyneuropathy, acute inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy;
  • peripheral nerve disease such as trigeminal neuralgia, facial paralysis, hemifacial spasm, vestibular neuronitis, glossopharyngeal neuralgia, mononeuropathy, brachial plexus neuralgia, polyneuropathy Mononeuropathy, polyneuropathy, acute inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy;
  • Spinal cord diseases such as myelitis, compressive myelopathy, subacute combined degeneration of the spinal cord, syringomyelia, spinal vasculopathy, spinal arachnoiditis, etc.;
  • Cerebrovascular diseases such as transient ischemic attack, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, intracranial venous system thrombosis, etc.;
  • Infectious diseases of the central nervous system such as meningitis, encephalitis, etc. caused by infections such as viruses, bacteria, fungi, or parasites, and lentiviral encephalitis caused by lentiviral infections;
  • Demyelinating diseases of the central nervous system such as multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, leukodystrophy, etc.;
  • Movement disorders such as Parkinson's disease, chorea, hepatolenticular degeneration, dystonia, essential tremor, tardive dyskinesia, etc.;
  • Headaches such as migraines, tension headaches, cluster headaches, etc.
  • Nervous system degenerative diseases such as motor neuron disease, Alzheimer's disease, Lewy body dementia, frontotemporal dementia, multiple system atrophy, etc.;
  • genetic diseases of the nervous system such as hereditary ataxia, hereditary spastic paraplegia, peroneal muscular atrophy, neurofibromatosis, tuberous sclerosis, cerebrofacial angiomatosis, etc.;
  • Nervous system developmental abnormalities such as congenital hydrocephalus, cerebral palsy, skull base depression, cerebellar subtonsillar disease, etc.;
  • Neuromuscular and muscle diseases such as myasthenia gravis, periodic paralysis, polymyositis, progressive muscular dystrophy, myotonic myopathies (myotonic dystrophy, congenital myotonia), metabolic myopathies (mitochondrial myopathy, lipid storage myopathy, glycogenosis), etc.;
  • Autonomic nervous system diseases such as Raynaud's disease, erythromelalgia, facial hemiatrophy, systemic autonomic insufficiency, spontaneous hyperhidrosis, progressive lipodystrophy, etc.;
  • Nervous system tumors such as glioma, lymphoma, meningioma, etc.:
  • Nervous system paraneoplastic syndromes such as paraneoplastic cerebellar degeneration, paraneoplastic encephalomyelitis, subacute necrotizing myelopathy, subacute motor neuron disease, paraneoplastic sensory neuron disease, etc.
  • neurodegenerative diseases lead to progressive loss of neuronal structure and function, including neuronal death and glial cell balance, leading to cognitive impairment such as dementia.
  • exemplary neurodegenerative diseases include Alzheimer's disease, Parkinson's disease (PD), Huntington's disease, early-onset AD or PD, and amyotrophic lateral sclerosis (ALS), among others.
  • the disease is a metabolic disease, such as diabetes, hyperlipidemia, gout, etc.
  • the metabolic disease-related targets are as described previously in this application.
  • the vector for the therapeutic nucleic acid in the aforementioned LNP is transposon DNA.
  • the LNP can contain chemotherapeutic drugs, which include but are not limited to cytotoxins, alkylating agents, podophyllotoxins, camptothecins, taxanes, antimetabolites, and antibiotics.
  • chemotherapeutic drugs include but are not limited to imidazole tetrazinone drugs, such as temozolomide; platinum drugs, such as oxaliplatin, cisplatin, carboplatin, nedaplatin, bicycline Platinum, lebaplatin, triplatinum tetranitrate, phenanthroplatin, picoplatin, saplatin; nitrosoureas, such as carmustine, cyclohexyl nitrosourea, methenyl nitrosourea, pyrimidine nitrosourea, carmustine , lomustine, fomustine, nimustine, ramustine, streptozotocin; camptothecin
  • the LNP can be encapsulated with cytotoxins, which include but are not limited to dolastatin, auristatin-like cytotoxic molecules, and maytansine.
  • Cytotoxic molecules; the DNA damaging agents include but are not limited to calicheamicins, duocarmycins, antromycin derivatives PBD, and camptothecin derivatives SN-38 .
  • the cytotoxin contained in the LNP is selected from the group consisting of amanitins, anthracyclines, baccatins, camptothecins, simadotin ( cemadotins), colchicines, colcimids, combretastatins, cryptophycins, discodermolides, docetaxel, doxorubicin (doxorubicin), echinomycins, eleuterobins, epothilones, estramustines, lexitropsins, maytansines , methotrexate, netropsins, puromycins, rhizoxins, taxanes, tubulysins, or periwinkle organisms Bases (vinca alkaloids).
  • the aforementioned fusion protein and cytotoxins are connected through a linker unit.
  • the cytotoxins include but are not limited to dolastatin and auristatin-type cytotoxic molecules, maytansine-type cytotoxic molecules. Cytotoxic molecules; the DNA damaging agents include but are not limited to calicheamicins, duocarmycins, antromycin derivatives PBD, and camptothecin derivatives SN-38.
  • the cytotoxin is selected from the group consisting of amanitins, anthracyclines, baccatins, camptothecins, cemadotins, colchicum colchicines, colcimids, combretastatins, cryptophycins, discodermolides, docetaxel, doxorubicin, echinacea Echinomycins, eleuterobins, epothilones, estramustines, lexitropsins, maytansines, methotrexate (methotrexate), netropsins, puromycins, rhizoxins, taxanes, tubulysins, or vinca alkaloids .
  • amanitins anthracyclines
  • baccatins camptothecins
  • cemadotins colchicum colchicines
  • colcimids colcimids
  • cryptophycins discodermolides
  • the aforementioned fusion protein is connected to a label, and the label is selected from the group consisting of iron oxide nanoparticles, UV-visible labels, near-infrared labels, luminescent groups, phosphorescent groups, magnetic spin resonance labels, photosensitizers, light A cleavable moiety, a chelating center, a heavy atom, a radioactive isotope, an isotope detectable spin resonance label, a paramagnetic moiety, a chromophore or a combination thereof; more preferably, the label is an iron oxide nanoparticle.
  • the aforementioned fusion proteins, nucleic acids, vectors, cells and/or prodrugs can be used to prepare drugs or kits for diagnosing, treating or preventing cancer or viral infection diseases.
  • the aforementioned diseases include cancer and viral infections.
  • the aforementioned cancers include, but are not limited to, leukemia, advanced adult cancer, pancreatic cancer, unresectable pancreatic cancer, colorectal cancer, metastatic colorectal cancer, ovarian cancer, triple negative breast cancer, hematopoietic/lymphoid cancer Cancer, colon cancer liver metastasis, small cell lung cancer, non-small cell lung cancer, B-cell lymphoma, relapsed or refractory B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large cell lymphoma, Relapsed or refractory diffuse large cell lymphoma, anaplastic large cell lymphoma, primary mediastinal B-cell lymphoma, recurrent mediastinal large B-cell lymphoma, refractory mediastinal large B-cell lymphoma, large B cellular lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma,
  • the viral infectious diseases include but are not limited to respiratory viral diseases, gastrointestinal viral diseases, liver viral diseases, skin and mucosal viral diseases, eye viral diseases, central nervous system viruses diseases, lymphocytic viral diseases, insect-borne viral diseases, lentiviral infectious diseases, etc.
  • respiratory viral diseases include rhinovirus, adenovirus, respiratory syncytial virus, parainfluenza virus, coronavirus, and the like. Infection; influenza; mumps, etc.;
  • gastrointestinal viral diseases include poliomyelitis; Cooksackie virus infection; ECHO virus infection; viral gastroenteritis: including rotavirus gastroenteritis, norovirus gastroenteritis, Adenovirus gastroenteritis, astrovirus gastroenteritis, coronavirus gastroenteritis and calicivirus gastroenteritis, etc.
  • liver viral diseases include viral hepatitis A, viral hepatitis B, viral hepatitis C, viral hepatitis D, viral hepatitis E, Epstein-Barr viral hepatitis, and cytomegalovirus Viral hepatitis, etc.
  • viral diseases of the skin and mucosa include measles, rubella, exanthema, varicella and shingles, smallpox, herpes simplex virus infection, rabies, foot and mouth disease, and the like.
  • ocular viral diseases include epidemic keratoconjunctivitis, follicular conjunctivitis, herpetic keratoconjunctivitis, and the like.
  • the viral diseases of the central nervous system include Japanese encephalitis, Western equine encephalitis, Eastern equine encephalitis, St. Louis encephalitis, Venezuelan equine encephalitis, Murray Valley encephalitis, California encephalitis inflammation, forest encephalitis and lymphocytic choriomeningitis.
  • lymphocytic viral diseases include infectious mononucleosis, cytomegalovirus infection, acquired immunodeficiency syndrome, and the like.
  • the insect-borne viral diseases include viral hemorrhagic fevers: including epidemic hemorrhagic fever, yellow fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, Argentine hemorrhagic fever, Venezuelan hemorrhagic fever, Lassa fever, Omsk hemorrhagic fever, Marburg disease and Ebola hemorrhagic fever, etc.; Dengue fever and dengue hemorrhagic fever; West Nile fever; Colorado tick-borne fever; sand fly fever, etc.
  • lentiviral infectious diseases include subacute sclerosing panencephalitis, kuru disease, progressive multifocal leukoencephalopathy, and subacute spongiform encephalopathy (corticostriatal spinal degeneration).
  • a transposon is a stretch of DNA that can insert itself into a location in the genome, e.g., a stretch of DNA that is capable of replicating itself and inserting a copy of it into the genome or that can be spliced out of a longer nucleic acid and inserted into the genome.
  • a stretch of DNA at another location in the genome e.g., transposons comprise DNA sequences composed of inverted repeats that flank the gene for transposition.
  • transposon refers to a nucleic acid segment that is recognized by a transposase or integrase and is an essential component of a functional nucleic acid-protein complex capable of transposition (ie, a transposome).
  • transposase refers to an enzyme that is a component of a functional nucleic acid-protein complex capable of transposition and mediates transposition.
  • transposase also refers to integrases derived from retrotransposons or retroviral origins.
  • Transposon complexes form between a transposase enzyme and a double-stranded DNA segment that contains specific binding sequences for the enzyme, termed "transposon ends.”
  • the sequence of the transposon binding site can be modified with other bases at certain positions without affecting the ability of the transposon complex to form a stable structure that can efficiently transpose into the target DNA.
  • chemotherapeutic drug refers to any chemical drug that has a therapeutic effect on a subject.
  • “Chemotherapeutic drugs” include but are not limited to anti-tumor drugs, and the chemotherapeutic drugs include but are not limited to dolastatin, auristatin-like cytotoxic molecules, and maytansine-like cytotoxic molecules;
  • the DNA damaging agents include but are not limited to calicheamicin, duocarmycin, antromycin derivative PBD, camptothecin derivative SN-38, amanitins ), anthracyclines, baccatins, camptothecins, cemadotins, colchicines, colcimids, combretastatin (combretastatins), cryptophycins, discodermolides, docetaxel, doxorubicin, echinomycins, eleuterobins, epothilones, estramustines, lexitropsins, may
  • Examples that can be cited include but are not limited to imidazole tetrazinone drugs, such as temozolomide; platinum drugs, such as oxaliplatin and cisplatin. , carboplatin, nedaplatin, bicycloplatin, lebaplatin, triplatinum tetranitrate, phenanthroplatin, picoplatin, satraplatin; nitrosoureas, such as carmustine, cyclohexyl nitrosourea, methylcyclonitrosourea, Pyrimidine nitrosoureas, carmustine, lomustine, fomustine, nimustine, ramustine, streptozotocin; camptothecins, such as camptothecin, hydroxycamptothecin , irinotecan, topotecan; vinblastines, such as vinorelbine, vinblastine, vincristine, vindesine, vinblastine; procarbazine;
  • cytotoxic includes any compound (such as a drug) that can kill T cells or reduce their activity.
  • the cytotoxins include but are not limited to dolastatin and auristatin-like cytotoxic molecules. Maytansine-like cytotoxic molecules; the DNA damaging agents include but are not limited to calicheamicin, duocarmycin, antromycin derivatives PBD, camptothecin derivatives SN-38, amanitins, anthracyclines, baccatins, camptothecins, cemadotins, colchicines, Colcimids, combretastatins, cryptophycins, discodermolides, docetaxel, doxorubicin, echinomycins , eleuterobins, epothilones, estramustines, lexitropsins, maytansines, methotrexate, spindle Netropsins, puromycins, rhizoxins, tax
  • linker refers to a peptide containing one or more amino acids, usually about 2 to 20 amino acids.
  • Peptide linkers are known in the art or described herein.
  • Suitable non-immunogenic linker peptides are, for example, (G4S)n, (SG4)n or G4(SG4)n peptide linkers, where "n” is usually a number between 1 and 10, usually between 1 and 4, in particular It's 2.
  • linker L1 appearing in this article includes but is not limited to GGGS (SEQ ID NO.20), GGGGS (SEQ ID NO.21), GGGGSGGGGS (SEQ ID NO.22), SGGGGSGGGG (SEQ ID NO.23), GGGGGSGGGGSSGGGGS (SEQ ID NO.24), GGGGSGGGGSGGGGS (SEQ ID NO.25), GGGGSGGGGSGGGG (SEQ ID NO.26), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO.27), GGGGSGGGGSGGGSGGGGS (SEQ ID NO.28), GSGSGSGS (SEQ ID NO.28).
  • linker L2 appearing in this article can be a polypeptide with a cysteine residue added to the C terminus of the aforementioned "linker L1", including but not limited to GGGSC (SEQ ID NO. 33), GGGGSGGGGSC (SEQ ID NO. .34), GGGGSGGGGSGGGGSGGGGSC (SEQ ID NO.35) and GGGGSGGGGGSGGGSGGGGSC (SEQ ID NO.36).
  • linker unit refers to a chemical structural segment or bond that is covalently linked to a ligand at one end and to a cytotoxic drug at the other end.
  • the linker unit used herein may include, but is not limited to: Maleimido-Caproyl-Valine-Citrulline-p-Aminobenzyloxy, mc-vc-pAB), maleimidocaproyl (mc), triglycyl peptide linker, 3-maleimido-propionic acid, Mal-di-EG-OPFP(perfluorophenyl 3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)propanoate), Mal-di-EG-Osu(2,5-dioxopyrrolidin-1-yl 3-(2-(2-(2,5-dioxo-2,5-dihydro-1H
  • label is used herein to refer to a compound or composition that is directly or indirectly coupled or fused to an agent, such as a nucleic acid probe or antibody, and that facilitates the detection of said agent coupled or fused thereto.
  • the label may itself be detectable (eg, a radioisotope label or a fluorescent label), or, in the case of an enzyme label, may catalyze a detectable chemical change in a substrate reactant or composition.
  • Labels may include iron oxide nanoparticles, UV-visible labels, near-infrared labels, luminescent groups, phosphorescent groups, magnetic spin resonance labels, photosensitizers, photocleavable moieties, chelating centers, heavy atoms, radioactive isotopes, isotopes Spin resonance tags, paramagnetic moieties, chromophores, or combinations thereof can be detected.
  • pharmaceutical formulation refers to a preparation in a form that permits the biological activity of the active ingredient contained therein to be effective, and which does not contain additional components that would have unacceptable toxicity to the subject to whom the formulation is to be administered.
  • pharmaceutically acceptable carrier refers to an ingredient of a pharmaceutical formulation, other than the active ingredient, which is not toxic to the subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • the choice of vector is determined in part by the specific cells and/or method of administration. Therefore, a variety of suitable formulations exist.
  • pharmaceutical compositions may contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixture thereof is typically present in an amount from about 0.0001% to about 2% by weight of the total composition.
  • Pharmaceutically acceptable carriers are generally nontoxic to the receptor at doses and concentrations used and include, but are not limited to: buffering agents such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives Agents (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens Esters, such as methyl or propyl paraben; catechol; resorcin; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues ) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, as
  • a buffering agent is included in the composition.
  • Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate and various other acids and salts.
  • a mixture of two or more buffers is used. The buffer or mixture thereof is typically present in an amount from about 0.001% to about 4% by weight of the total composition. Methods of preparing pharmaceutical compositions for administration are known.
  • Formulations may include aqueous solutions.
  • the formulation or composition may also contain more than one active ingredient useful for the particular indication, disease or condition being treated with the cell, preferably those with activities that are complementary to the cell, where the respective activities do not adversely affect one another.
  • active ingredients are suitably present in combination in amounts effective for the intended purpose.
  • the pharmaceutical compositions further comprise other pharmaceutically active agents or drugs, such as chemotherapeutic agents such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil , gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine and/or vincristine.
  • a pharmaceutical composition contains an amount of cells effective to treat or prevent a disease or disorder, eg, a therapeutically effective amount or a prophylactically effective amount.
  • therapeutic or prophylactic efficacy is monitored by periodic assessment of treated subjects.
  • the desired dose can be delivered by administering the cells as a single bolus, as multiple bolus, or as a continuous infusion.
  • Cells and compositions can be administered using standard administration techniques, formulations and/or devices. Administration of cells can be autologous or allogeneic. For example, immune response cells or progenitor cells can be obtained from one subject and administered to the same subject or to a different, compatible subject. Peripheral blood-derived immune response cells or progeny thereof (eg, derived in vivo, ex vivo, or in vitro) can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When therapeutic compositions (eg, pharmaceutical compositions containing genetically modified immune response cells) are administered, they are typically formulated in unit dose injectable forms (solutions, suspensions, emulsions).
  • therapeutic compositions eg, pharmaceutical compositions containing genetically modified immune response cells
  • Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration.
  • the cell population is administered parenterally.
  • parenteral as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal and intraperitoneal administration.
  • cells are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
  • the compositions are provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in certain aspects can be buffered to a selected pH.
  • sterile liquid preparations such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in certain aspects can be buffered to a selected pH.
  • Liquid preparations are generally easier to prepare than gels, other viscous compositions and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide longer contact time with specific tissues.
  • Liquid or viscous compositions may include a carrier, which may be a solvent or dispersion medium, including, for example, water, saline, phosphate buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.
  • a carrier which may be a solvent or dispersion medium, including, for example, water, saline, phosphate buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.
  • Sterile injectable solutions can be prepared by introducing the cells into a solvent, e.g., mixed with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, and the like.
  • a suitable carrier e.g., sterile water, physiological saline, glucose, dextrose, and the like.
  • the compositions may contain auxiliary substances such as wetting, dispersing or emulsifying agents (e.g. methylcellulose), pH buffers, gelling or thickening additives, preservatives, flavorings and/or color, depending on Routes of Administration and Desired Preparations.
  • auxiliary substances such as wetting, dispersing or emulsifying agents (e.g. methylcellulose), pH buffers, gelling or thickening additives, preservatives, flavorings and/or color, depending on Routes of Administration and Desired Preparations.
  • preservatives e.g. methylcellulose
  • additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents and buffers. Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol and sorbic acid. Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents which delay absorption such as aluminum monostearate and gelatin.
  • Formulations for in vivo administration are generally sterile. Sterility can be easily achieved by, for example, filtration through a sterile membrane.
  • cells, populations and compositions are administered to a subject or patient suffering from a particular disease or condition to be treated, such as by adoptive cell therapy (eg, adoptive T cell therapy).
  • adoptive cell therapy eg, adoptive T cell therapy
  • cells and compositions e.g., engineered compositions and production end products after incubation and/or other processing steps
  • methods thereby treat, eg, one or more symptoms of a disease or disorder, eg, by reducing tumor burden in a cancer that expresses an antigen recognized by engineered T cells.
  • Cell administration methods for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions.
  • methods of adoptive T cell therapy are described 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) .
  • a "subject" as used herein is a mammal, such as a human or other animal, and is typically a human.
  • a subject such as a patient, to which a cell, cell population or composition is administered is a mammal, typically a primate such as a human.
  • the primate is a monkey or ape.
  • Subjects may be male or female and of any appropriate age, including infants, juveniles, adolescents, adults, and geriatric subjects.
  • the subject is a non-primate mammal, such as a rodent.
  • Treatment as used herein (and its grammatical variations such as “treatment” or “treatment”) means the complete or partial amelioration or alleviation of a disease or condition or disorder, or the symptoms, adverse effects or consequences associated therewith or Phenotype. Desired therapeutic effects include, but are not limited to, prevention of the occurrence or recurrence of disease, alleviation of symptoms, alleviation of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, alleviation or reduction of disease status, and alleviation or improvement of prognosis. These terms do not imply complete cure of a disease or complete elimination of any symptoms or effect on all symptoms or consequences.
  • delaying the development of a disease means delaying, hindering, slowing, retarding, stabilizing, inhibiting and/or delaying the development of a disease (eg, cancer).
  • This delay can be of varying lengths of time, depending on the history of the disease and/or the individual being treated. It will be apparent to those skilled in the art that a sufficient or significant delay may actually include prevention because the individual does not develop the disease. For example, the development of advanced cancer such as metastasis may be delayed.
  • Prevention includes providing prevention against the occurrence or recurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease.
  • cells and compositions are provided for delaying the development of a disease or slowing the progression of a disease.
  • “Inhibiting” function or activity as used herein means reducing function or activity when compared to the same conditions (other than the condition or parameter of interest), or when compared to another condition. For example, a cell that inhibits tumor growth reduces the growth rate of a tumor compared to the growth rate of the tumor in the absence of the cell.
  • an "effective amount" of an agent means effective in achieving the desired result at the dose/amount and time period necessary For example, the amount of therapeutic or preventive results.
  • a "therapeutically effective amount" of an agent is effective, at the dosage and for the time period necessary, to achieve the desired therapeutic effect (e.g., for treatment of a disease, condition, or disorder, and/or the pharmacokinetics of the treatment) or pharmacodynamic effect) amount.
  • the therapeutically effective amount may vary depending on factors such as the disease state, the age, sex, and weight of the subject, and the cell population administered.
  • provided methods include administering cells and/or compositions in an effective amount, such as a therapeutically effective amount.
  • Preventatively effective amount refers to the amount effective to achieve the desired preventive results at the necessary dosage and time period. Typically, but not necessarily, the prophylactically effective amount will be less than the therapeutically effective amount because the prophylactic dose is administered in the subject prior to or early in the course of the disease. At lower tumor burdens, the prophylactically effective amount will in some aspects be higher than the therapeutically effective amount.
  • cells or individual populations or subtypes of cells are administered to a subject in the range of about one million to about one hundred billion cells, such as 1 million to about 50 billion cells (e.g., about 500 Ten thousand 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 any two of the above range defined by values), for example, about 10 million to about 100 billion cells (for example, about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 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, from about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells cells, about 800 million cells, about 900 million cells, about 3 billion cells
  • the dose of total cells and/or the dose of individual subpopulations of cells is between at or about 10 4 cells/kilogram (kg) body weight to at or about 10 9 cells/kilogram (kg) body weight Within the range, for example, between 10 5 and 10 6 cells/kg body weight, for example, at least or at least about or at or about 1 ⁇ 10 5 cells/kg, 1.5 ⁇ 10 5 cells/kg, 2 ⁇ 10 5 cells/kg or 1 ⁇ 10 6 cells/kg body weight.
  • the cells are administered at or within a range of between or about 10 4 and at or about 10 9 T cells/kilogram (kg) of body weight, e.g., between 10 5 and 10 6 T cells /kg body weight, such as at least or at least about or at or about 1 ⁇ 10 5 T cells/kg, 1.5 ⁇ 10 5 T cells/kg, 2 ⁇ 10 5 T cells/kg, or 1 ⁇ 10 6 T cells/kg body weight.
  • body weight e.g., between 10 5 and 10 6 T cells /kg body weight, such as at least or at least about or at or about 1 ⁇ 10 5 T cells/kg, 1.5 ⁇ 10 5 T cells/kg, 2 ⁇ 10 5 T cells/kg, or 1 ⁇ 10 6 T cells/kg body weight.
  • the cells may be administered by any suitable means, such as by bolus injection, by injection, such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, Intrachoroidal injection, intracameral injection, subperineal injection, subconjunctival injection, sub-Tenon injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery.
  • they are administered parenterally, intrapulmonary, and intranasally, and if local treatment is desired, intralesional administration.
  • Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • a given dose is administered by administering the cells as a single bolus. In some embodiments, it is administered by administering the cells as multiple bolus injections, for example, over a period of no more than 3 days, or by continuous infusion of the administered cells.
  • repeated dosing methods are provided wherein a first dose of cells is administered followed by one or more second consecutive doses.
  • multiple doses of cells are typically timed and sized to increase the efficacy and/or activity and/or function of antigen-expressing T cells (eg, CAR-expressing T cells).
  • repeated dosing reduces the down-regulation or inhibitory activity that can occur when inhibitory immune molecules, such as PD-1 and/or PD-L1, are up-regulated on antigen-expressing, eg, CAR-expressing, T cells.
  • Methods include administering a first dose, usually followed by one or more consecutive doses, with a specified time frame between doses.
  • administration of a given "dose” includes administration of a given amount or number of cells as a single composition and/or as a single uninterrupted administration (e.g., as a single injection or continuous infusion), and also includes the administration of a given amount or number of cells as divided doses in multiple individual compositions or infusions over a specified period of time (not to exceed 3 days).
  • the first or sequential dose is a single or sequential administration of a specified number of cells administered or initiated at a single time point.
  • the first or subsequent doses are administered as multiple injections or infusions over a period of not more than three days, such as three or two days, once daily, or as multiple infusions over a single day. .
  • Example 1 Fusion protein targeting antigen to promote antigen endocytosis
  • This embodiment designs a fusion protein that promotes endocytosis of targeted antigens. On this basis, this embodiment designs a fusion protein that targets TCR and promotes TCR endocytosis.
  • FIG. 1A The basic structure of the fusion protein is shown in Figure 1A, which includes a targeting domain, a linker, and a trimer domain.
  • the protein structural form of the fusion protein may be a monomer, a dimer (the targeting domain forms a heterodimer), a dimer (the targeting domain forms a homodimer) or a trimer. body. As shown in Figure 1G.
  • this embodiment designs a fusion protein BMA-STII-VXT that targets TCR and promotes TCR endocytosis (the amino acid sequence of the fusion protein BMA-STII-VXT is shown in SEQ ID NO. 36), which includes A single-chain antibody fragment (scFv) targeting the TCR, Strep tag II tag, Glycine-Serine linker, and trimer domain (VXT) from collagen XV is shown in Figure 1B.
  • scFv single-chain antibody fragment
  • Strep tag II tag Strep tag II tag
  • Glycine-Serine linker Glycine-Serine linker
  • VXT trimer domain
  • the single-chain antibody targeting TCR ⁇ is the antibody BMA031 that specifically binds to the constant epitope of the TCR ⁇ /CD3 complex:
  • the amino acid sequence of the heavy chain variable region of the TCR-targeting single-chain antibody BMA031 is shown in SEQ ID NO.7
  • STII Tetrachloro-Pro-Gln-Phe-Glu-Lys
  • STII Tetrachloro-acid Strep Tag II tag.
  • the binding affinity of Strep-tag II to Strep-Tactin is nearly 100 times higher than that of streptavidin. Therefore, Strep-Tactin can be used for purification;
  • VXT is a component that promotes trimer formation, which is derived from the trimer domain of human type XV collagen (the amino acid sequence of VXT is shown in SEQ ID NO. 18).
  • the VXT domain contains four ⁇ -sheet structures and ⁇ -helices, which can form a triple-helix structure.
  • the nucleic acid sequence encoding a fusion protein that targets TCR and promotes TCR endocytosis is cloned into a eukaryotic expression plasmid, and the plasmid is transfected into the engineering cell line Expi293 (purchased from ThermoFisher, Cat. No.: A14635) and cultured for 7 days, The culture supernatant was collected, purified by StrepTactin using a purification column Starm Streptacitin Beeds (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd., Cat. No. SA092005), and eluted with Biotin, and then separated by SDS-PAGE gel electrophoresis. The results It shows that in the reduced state, the expressed fusion protein is a monomer; in the non-reduced state, the expressed fusion protein is a dimer structure.
  • this example designed a fusion protein BMC-STII-VXT that targets CD3 and promotes CD3 endocytosis, which includes a single-chain antibody fragment BMC (scFv) targeting CD3, a Strep tag II tag, and a glyserine hinge. region (Glycine-Serine linker) and the trimer domain (VXT) from collagen XV, as shown in Figure 1C.
  • scFv single-chain antibody fragment BMC
  • Strep tag II tag Strep tag II tag
  • VXT trimer domain
  • the single-chain antibody targeting CD3 is the antibody BMC that specifically binds to the CD3 receptor protein:
  • the amino acid sequence of the heavy chain variable region of the CD3-targeting single-chain antibody BMC is shown in SEQ ID NO. 15:
  • STII Tetrachloro-Pro-Gln-Phe-Glu-Lys
  • STII Tetrachloro-acid Strep Tag II tag.
  • the binding affinity of Strep-tag II to Strep-Tactin is nearly 100 times higher than that of streptavidin. Therefore, Strep-Tactin can be used for purification;
  • VXT is a component that promotes trimer formation, which is derived from the trimer domain of human type XV collagen (the amino acid sequence of VXT is shown in SEQ ID NO. 18).
  • the VXT domain contains four ⁇ -sheet structures and ⁇ -helices, which can form a triple-helix structure.
  • this example designed a fusion protein BMD-STII-VXT that targets CD5 and promotes CD5 endocytosis, which includes a single-chain antibody fragment BMD (scFv) targeting CD5, a Strep tag II tag, and a glyserine hinge. region (Glycine-Serine linker) and the trimer domain (VXT) from collagen XV, as shown in Figure 1D.
  • scFv single-chain antibody fragment BMD
  • Strep tag II tag Strep tag II tag
  • VXT trimer domain
  • the single-chain antibody targeting CD5 is an antibody BMD that specifically binds to the CD5 receptor protein:
  • the amino acid sequence of the heavy chain variable region of the CD5-targeting single-chain antibody BMD is shown in SEQ ID NO. 221:
  • STII Tetrachloro-Pro-Gln-Phe-Glu-Lys
  • STII Tetrachloro-acid Strep Tag II tag.
  • the binding affinity of Strep-tag II to Strep-Tactin is nearly 100 times higher than that of streptavidin. Therefore, Strep-Tactin can be used for purification;
  • VXT is a component that promotes trimer formation, which is derived from the trimer domain of human type XV collagen (the amino acid sequence of VXT is shown in SEQ ID NO. 18).
  • the VXT domain contains four ⁇ -sheet structures and ⁇ -helices, which can form a triple-helix structure.
  • this example designed a fusion protein BME-STII-VXT that targets CD7 and promotes CD7 endocytosis, which includes a single-chain antibody fragment BME (scFv) targeting CD7, a Strep tag II tag, and a glyserine hinge. region (Glycine-Serine linker) and the trimer domain (VXT) from collagen XV, as shown in Figure 1E.
  • scFv single-chain antibody fragment BME
  • Strep tag II tag Strep tag II tag
  • VXT trimer domain
  • the single-chain antibody targeting CD7 is the antibody BME that specifically binds to the CD7 receptor protein:
  • the amino acid sequence of the heavy chain variable region of the CD7-targeting single-chain antibody BME is shown in SEQ ID NO. 229:
  • STII Tetrachloro-Pro-Gln-Phe-Glu-Lys
  • STII Tetrachloro-acid Strep Tag II tag.
  • the binding affinity of Strep-tag II to Strep-Tactin is nearly 100 times higher than that of streptavidin. Therefore, Strep-Tactin can be used for purification;
  • VXT is a component that promotes trimer formation, which is derived from the trimer domain of human type XV collagen (the amino acid sequence of VXT is shown in SEQ ID NO. 18).
  • the VXT domain contains four ⁇ -sheet structures and ⁇ -helices, which can form a triple-helix structure.
  • this example designed a fusion protein BMF-STII-VXT that targets CD4 and promotes CD4 endocytosis, which includes a single-chain antibody fragment BMF (scFv) targeting CD4, a Strep tag II tag, and a glyserine hinge. region (Glycine-Serine linker) and the trimer domain (VXT) from collagen XV, as shown in Figure 1F.
  • scFv single-chain antibody fragment BMF
  • Strep tag II tag Strep tag II tag
  • VXT trimer domain
  • the single-chain antibody targeting CD4 is the antibody BMF that specifically binds to the CD4 receptor protein:
  • the amino acid sequence of the heavy chain variable region of the CD4-targeting single-chain antibody BMF is shown in SEQ ID NO. 237:
  • STII Tetrachloro-Pro-Gln-Phe-Glu-Lys
  • STII Tetrachloro-acid Strep Tag II tag.
  • the binding affinity of Strep-tag II to Strep-Tactin is nearly 100 times higher than that of streptavidin. Therefore, Strep-Tactin can be used for purification;
  • VXT is a component that promotes trimer formation, which is derived from the trimer domain of human type XV collagen (the amino acid sequence of VXT is shown in SEQ ID NO. 18).
  • the VXT domain contains four ⁇ -sheet structures and ⁇ -helices, which can form a triple-helix structure.
  • this example designed a fusion protein BMF-STII-VXT that targets CD20 and promotes CD20 endocytosis, which includes a single-chain antibody fragment BMG (scFv) targeting CD20, a Strep tag II tag, and a glyserine hinge. region (Glycine-Serine linker) and the trimer domain (VXT) from collagen XV, as shown in Figure 1G.
  • scFv single-chain antibody fragment BMG
  • Strep tag II tag Strep tag II tag
  • VXT trimer domain
  • the single-chain antibody targeting CD20 is the antibody BMG that specifically binds to the CD20 receptor protein:
  • the amino acid sequence of the heavy chain variable region of the CD20-targeting single-chain antibody BMF is shown in SEQ ID NO. 245:
  • STII Tetrachloro-Pro-Gln-Phe-Glu-Lys
  • STII Tetrachloro-acid Strep Tag II tag.
  • the binding affinity of Strep-tag II to Strep-Tactin is nearly 100 times higher than that of streptavidin. Therefore, Strep-Tactin can be used for purification;
  • VXT is a component that promotes trimer formation, which is derived from the trimer domain of human type XV collagen (the amino acid sequence of VXT is shown in SEQ ID NO. 18).
  • the VXT domain contains four ⁇ -sheet structures and ⁇ -helices, which can form a triple-helix structure.
  • this example designed a fusion protein BMF-STII-VXT that targets CD22 and promotes CD22 endocytosis, which includes a single-chain antibody fragment BMH (scFv) targeting CD22, a Strep tag II tag, and a glyserine hinge. region (Glycine-Serine linker) and the trimer domain (VXT) from collagen XV, as shown in Figure 1H.
  • scFv single-chain antibody fragment BMH
  • Strep tag II tag Strep tag II tag
  • VXT trimer domain
  • the single-chain antibody targeting CD22 is the antibody BMH that specifically binds to the CD22 receptor protein:
  • the amino acid sequence of the heavy chain variable region of the CD22-targeting single-chain antibody BMF is shown in SEQ ID NO. 253:
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO. 254:
  • STII Tetrachloro-Pro-Gln-Phe-Glu-Lys
  • STII Tetrachloro-acid Strep Tag II tag.
  • the binding affinity of Strep-tag II to Strep-Tactin is nearly 100 times higher than that of streptavidin. Therefore, Strep-Tactin can be used for purification;
  • VXT is a component that promotes trimer formation, which Trimeric domain from human type XV collagen (the amino acid sequence of VXT is shown in SEQ ID NO. 18).
  • the VXT domain contains four ⁇ -sheet structures and ⁇ -helices, which can form a triple-helix structure.
  • this example designed a fusion protein BMF-STII-VXT that targets CD64 and promotes CD64 endocytosis, which includes a single-chain antibody fragment BMI (scFv) targeting CD64, a Strep tag II tag, and a glyserine hinge. region (Glycine-Serine linker) and the trimer domain (VXT) from collagen XV, as shown in Figure 1I.
  • scFv single-chain antibody fragment BMI
  • Strep tag II tag Strep tag II tag
  • VXT trimer domain
  • the single-chain antibody targeting CD64 is the antibody BMI that specifically binds to the CD64 receptor protein:
  • the amino acid sequence of the heavy chain variable region of the CD64-targeting single-chain antibody BMF is shown in SEQ ID NO. 261:
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO. 262:
  • STII Tetrachloro-Pro-Gln-Phe-Glu-Lys
  • STII Tetrachloro-acid Strep Tag II tag.
  • the binding affinity of Strep-tag II to Strep-Tactin is nearly 100 times higher than that of streptavidin. Therefore, Strep-Tactin can be used for purification;
  • VXT is a component that promotes trimer formation, which is derived from the trimer domain of human type XV collagen (the amino acid sequence of VXT is shown in SEQ ID NO. 18).
  • the VXT domain contains four ⁇ -sheet structures and ⁇ -helices, which can form a triple-helix structure.
  • TCR-targeting fusion protein specifically binds to T cells
  • Peripheral blood mononuclear cells were isolated from whole blood donated by healthy individuals. Mononuclear cells were isolated using lymphocyte density gradient separation. Isolated mononuclear cells were activated with antibodies against CD3 and CD28 to obtain T cells.
  • the BMA-STII-VXT fusion protein prepared in Example 1 was modified with Biotin, then incubated with T cells, and then flow cytometry was performed on the T cells. A total of 5 sets of experiments were conducted to detect the BMA-STII-VXT fusion protein. The ability to bind to T cells, the experimental process is as follows:
  • B.0.5M T cells are first labeled with 1 ⁇ L of BMA-STII-VXT-Biotin for 30 minutes, then eluted, and then stained with CD8- Pacific blue (ThermoFisher, Cat. No.: MHCD0828), Streptavidin-PE (Biolegend, Cat. No.: 405204)15 minutes, flow cytometry was performed after elution (the flow cytometry results are shown in Figure 2B);
  • T cells were first labeled with 3 ⁇ L of BMA-STII-VXT-Biotin for 30 minutes, then eluted, and then stained with CD8- Pacific blue (ThermoFisher, Cat. No.: MHCD0828), Streptavidin-PE (Biolegend, Cat. No.: 405204) 15 minutes, flow cytometry was performed after elution (the flow cytometry results are shown in Figure 2C);
  • T cells D.0.5M T cells were first labeled with 3 ⁇ L of BMA-STII-VXT-Biotin for 30 minutes, then eluted, and then used CD8- Pacific blue (ThermoFisher, Catalog No.: MHCD0828), TCR- ⁇ -APC-Cy7 (biolegend, Catalog No.: 109220) for 15 minutes, and flow cytometry was performed after elution (the flow cytometry results are shown in Figure 2D);
  • E.0.5M T cells are first labeled with 3 ⁇ L of BMA-STII-VXT-Biotin for 30 minutes, and then washed with CD8- Pacific blue (ThermoFisher, Cat. No.: MHCD0828), Streptavidin-PE (Biolegend, Cat. No.: 405204), TCR - ⁇ -APC-Cy7 (biolegend, Cat. No.: 109220) was stained for 15 minutes, and flow cytometry was performed after elution (the flow cytometry results are shown in Figure 2E).
  • Example 3 Fusion protein targeting TCR promotes TCR endocytosis
  • T cells were simultaneously labeled with TCR- ⁇ antibodies and incubated, and then flow cytometric detection was performed.
  • the preparation method of T cells is as shown in Example 2. A total of four sets of experiments were conducted, and the experimental processes are as follows:
  • TCR- ⁇ -APC-Cy7 biolegend, Cat. No.: 109220
  • TCR-ab flow cytometric detection
  • B. Use BMA-STII-VXT-Biotin to label T cells for 30 minutes, incubate for 30 minutes after elution, and then use Streptavidin-PE (biolegend, Cat. No.: 405204) and TCR- ⁇ -APC-Cy7 (biolegend, Cat. No.: 109220) , stain for 15 minutes, and perform flow cytometry after elution; the results show that BMA-STII-VXT can completely shield TCR- ⁇ antibodies and compete for binding to T cells (flow cytometry results are shown in Figure 3B);
  • T cells were labeled with BMA-STII-VXT-Biotin for 30 minutes, incubated for 6 hours after elution, and then stained with Streptavidin-PE (biolegend, Cat. No.: 405204) and TCR- ⁇ -APC-Cy7 (biolegend, Cat. No.: 109220) for 15 minutes.
  • Streptavidin-PE biolegend, Cat. No.: 405204
  • TCR- ⁇ -APC-Cy7 biolegend, Cat. No.: 109220
  • BMA-STII-VXT can completely shield TCR- ⁇ antibodies so that TCR- ⁇ antibodies cannot bind to T cells. It can also promote the TCR bound to BMA-STII-VXT to be endocytosed into T cells without binding to BMA. -The TCR bound by STII-VXT returns to the T cell surface after a period of time, so that it can bind to anti-TCR ⁇ antibodies.
  • Example 4 Drug delivery using TCR-targeting fusion protein conjugated with LNP
  • the LNP By connecting LNP directly or indirectly through a linker to a TCR-targeting fusion protein, the LNP contains nucleic acid that expresses specific functional proteins to transform T cells into CAR-T cells, thereby achieving therapeutic effects on tumors or viral infections. .
  • the LNP By connecting LNP directly or indirectly through a linker to a TCR-targeting fusion protein, the LNP contains nucleic acids that express specific functional proteins to transform T cells into CNK-UT cells, thereby achieving therapeutic effects on tumors or viral infections. .
  • the LNP By connecting LNP directly or indirectly through a linker to a TCR-targeting fusion protein, the LNP contains nucleic acids that express specific functional proteins to transform T cells into expressing chimeric protein constructs TPD, thereby achieving the goal of treating tumors or viral infections. therapeutic effect.
  • the biologically active nucleic acid contained in the LNP is a nucleic acid encoding a transposon, and the nucleic acid is included in the aforementioned chimeric protein receptor (CAR).
  • CAR chimeric protein receptor
  • CNK-UT complex or chimeric protein construct are embedded with inverted repeat sequence nucleic acid and transposase-encoding nucleic acid at both ends, allowing T cells to express specific proteins (the process is shown in Figure 5), thereby achieving tumor targeting or the therapeutic effect of viral infections.
  • LNP By connecting LNP to TCR-targeting fusion proteins directly or indirectly through a linker (the structure is shown in Figure 6), targeted delivery of chemotherapy drugs into T cells can achieve therapeutic effects on tumors or viral infections.
  • the small molecule drugs can be targeted and delivered into T cells to directly achieve the function of killing T cells and can be used to treat leukemia, Adult advanced cancer, pancreatic cancer, unresectable pancreatic cancer, colorectal cancer, metastatic colorectal cancer, ovarian cancer, triple negative breast cancer, hematopoietic/lymphoma, colon cancer liver metastasis, small cell lung cancer, non-small cell lung cancer, B-cell lymphoma, relapsed or refractory B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large cell lymphoma, relapsed or refractory diffuse large cell lymphoma, anaplastic large cell lymphoma cell lymphoma, primary mediastinal B-cell lymphoma, relapsed mediastinal large B-cell lymphoma, refractory
  • Example 6 Labeling T cells using fusion proteins targeting TCR
  • Iron oxide nanoparticles are connected to the TCR-targeting fusion protein to label T cells, so that in vitro detection and monitoring of T cells reinfused into the body can be achieved, so as to understand the migration and distribution of T cells and evaluate T cells.
  • Cell functions such as targeting and persistence.

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Abstract

La présente invention concerne une protéine de fusion ciblant des protéines de récepteur de membrane cellulaire et leur utilisation. La protéine de fusion ciblant les protéines de récepteur de membrane cellulaire peut favoriser l'endocytose de protéines de récepteur de membrane qui est obtenue au moyen du mécanisme d'endocytose dépendant de la clathrine (CDE).
PCT/CN2023/095988 2022-05-25 2023-05-24 Protéine de fusion ciblant des protéines de récepteur de membrane cellulaire et son utilisation WO2023227018A1 (fr)

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