WO2023227018A1 - Fusion protein targeting cell membrane receptor proteins and use thereof - Google Patents

Fusion protein targeting cell membrane receptor proteins and use thereof Download PDF

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Publication number
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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French (fr)
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.

Abstract

Provided in the present disclosure are fusion protein targeting cell membrane receptor proteins and the use thereof. The fusion protein targeting the cell membrane receptor proteins can promote the endocytosis of membrane receptor proteins which is achieved by means of the clathrin-dependent endocytosis (CDE) mechanism.

Description

靶向细胞膜受体蛋白的融合蛋白及其应用Fusion proteins targeting cell membrane receptor proteins and their applications 技术领域Technical field
本公开涉及治疗领域，更具体地，涉及一种靶向细胞膜受体蛋白的融合蛋白及其应用。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.
背景技术Background technique
内吞(Endocytosis)是细胞运行过程中通过细胞膜吞噬(engulfment)的途径将小分子或底物转移到细胞的机制。内吞通常分成吞噬作用(phagocytosis)和胞饮作用(pinocytosis)，其中吞噬作用指的是内吞较大的实颗粒(直径0.5–10μm)，例如病原微生物。胞饮作用是通过小的内吞小泡内吞各种脂质。病原体或配体的内吞通过结合细胞膜表面，通过受体依赖性或受体不依赖性机制，形成内吞体(Phagosome)或内吞小泡(endocytic vescle)。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 μm 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.
膜受体和胞外分子结合负责传导胞外信号到细胞内。研究表明，一些细胞膜受体与配体结合形成复合物大部分通过网格蛋白依赖性(Clathrin-dependent endocytosis)内吞机制内化和转移到胞质内或核内进行信号传递或降解(Aguilar RC et al.,2005)。如T细胞(抗原)受体(T cell receptor，TCR)，络氨酸受体激酶(receptor tyrosine kinases，RTKs)和G-蛋白藕连受体(G-protein coupled receptors,GPCRs)能通过直接与适配体蛋白AP2,epsin或intersectin结合，通过网格蛋白(clathrin)依赖性内吞机制实现内吞(Kourouniotis G et al.,2016；Poole DP et al.,2016)。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). For example, T cell (antigen) receptors (TCR), tyrosine receptor kinases (RTKs) and G-protein coupled receptors (GPCRs) can interact directly with The aptamer protein AP2, epsin or intersectin binds to achieve endocytosis through a clathrin-dependent endocytosis mechanism (Kourouniotis G et al., 2016; Poole DP et al., 2016).
T细胞(抗原)受体(TCR)是位于T细胞表面的一种异二聚体蛋白，是T细胞表面特异性识别抗原和介导免疫应答的分子。由于细胞发育过程中TCR存在复杂的V(D)J重排机制，导致TCR基因序列多样性极为丰富，因此TCR具有非常高的多样性，是人类基因组中多态性最高的区域之一。TCR由两条多肽链组成(α/β或γ/δ)，每条链分为恒定区和可变区，且每条链包含及个高度可变区CDR1(complementarity－determining regions，CDR，互补决定簇1)、CDR2、CDR3。CDR1和CDR2相对保守，负责识别MHC；CDR3是TCR直接接触抗原肽的区域，对TCR与抗原肽-MHC复合体的互作起到决定性作用，且CDR3是可变程度最高的区域，很大程度上决定了TCR的多样性。T cell (antigen) receptor (TCR) is a heterodimeric protein located on the surface of T cells. It is a molecule on the surface of T cells that specifically recognizes antigens and mediates immune responses. Due to the complex V(D)J rearrangement mechanism of TCR during cell development, the diversity of TCR gene sequences is extremely rich. Therefore, TCR has very high diversity and is one of the most polymorphic regions in the human genome. TCR is composed of two polypeptide chains (α/β or γ/δ). 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.
静息期的T细胞，TCR会被持续的内吞并重新表达在细胞表面。但当TCR和pMHC结合，会形成pMHC/TCR/CD3的免疫复合物突触(Immune Synapse)，TCR内吞会迅速增加并下调在细胞表面的表达，减少再循环，加快TCR的降解，同时激活T细胞内部的信号通路,刺激T细胞扩增或杀伤效应。在TCR激活过程中，结合的TCR和周围不结合的TCR都能通过Clathrin依赖性或不依赖性的内吞机制(endocytosis)实现内化。被内化的结合的TCR/CD3复合物会被降解，但周围的TCR/CD3复合物大部分会重新循环迁徙到细胞膜表面的免疫突触复合物(immunological synapse)。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抗体刺激也可以导致TCR内吞，这个过程也是Clathrin介导的内吞机制(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.)。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是促三聚体结构域来自人源XV型胶原蛋白的三聚体结构域。胶原蛋白分子是细胞外基质中的主要成分，对细胞的粘附、运动、迁徙等活动起到重要作用。XVT结构来源于人源XV型或XVIII型胶原蛋白的三聚体结构域，这个结构域包含4个β-折叠结构和α-螺旋,可以形成三螺旋的结构。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 β-sheet structures and α-helices, which can form a triple helix structure.
发明内容Contents of the invention
本公开提供了一种通过设计和表达包含靶向TCR的结构域与促三聚体结构域的重组蛋白，靶向结合T细胞表面的TCR结构，从而实现TCR结构聚集，形成免疫突触，从而诱导TCR结构的内吞，实现靶向T细胞定向标记、投递药物和核酸或实现检测或治疗的功能。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.
本公开还提供了一种通过设计和表达包含靶向CD3的结构域与促三聚体结构域的重组蛋白，靶向结合T细胞表面的CD3，从而实现CD3聚集，诱导CD3的内吞，实现靶向T细胞定向标记、投递药物和核酸或实现检测或治疗的功能。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是T细胞和部分B1a类B细胞膜表面恒定表达的清道夫受体家族成员蛋白。通过抗CD5mAb处理T细胞和B细胞，CD5能被迅速内吞(Xianghuai Lu et al.,2002)。靶向CD5的LNP能高效递送CAR mRNA进入T细胞，并在体内改造T细胞成FAP CAR-T细胞，能清除在心衰过程中激活的成纤维细胞，达到治疗心脏纤维化的目的(Rurik JG et al.,CAR T cells produced in vivo to treat cardiac injury.Science.2022Jan 7；375(6576):91-96.)。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.).
本公开还提供了一种通过设计和表达包含靶向CD5的结构域与促三聚体结构域的重组蛋白，靶向结合T细胞表面的CD5，从而实现CD5聚集，诱导CD5的内吞，实现靶向T细胞定向标记、投递药物和核酸或实现检测或治疗的功能。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是40KD单域Ig超家族分子穿膜糖蛋白，密集表达在人分化成熟的T细胞和NK细胞表面及其前体细胞上(BF Haynes et al.,1979，H Rabinowich et al.,1994)。CD7作为T细胞的共刺激因子，通过结合其配体 K12/SECTM,在T细胞激活过程中发挥重要作用(Lyman SD et al.,2000)。CD7高表达在所有T细胞急性淋巴细胞白血病细胞上。由于CD7与其配体、抗体结合后能迅速内吞，而不从细胞表面切割丢失。因此，CD7作为治疗T淋巴细胞白血病的ADC药物的重要靶点。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.
本公开还提供了一种通过设计和表达包含靶向CD7的结构域与促三聚体结构域的重组蛋白，靶向结合T细胞表面的CD7，从而实现CD7聚集，诱导CD7的内吞，实现靶向T细胞定向标记、投递药物和核酸或实现检测或治疗的功能。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抗原(也被称作人B淋巴细胞限制的分化抗原，Bp35)是位于前-B与成熟B淋巴细胞上的疏水跨膜蛋白质，具有分子量大约35kD(J.Biol.Chem.264(19):11282-11287(1989)；Einfeld et al.EMBO J.7(3):711-717(1988))。该抗原也在超过90％的B细胞非何杰金氏淋巴瘤(NHL)上表达(Blood63(6):1424-1433(1984))，但是在造血干细胞、原B细胞、正常浆细胞或者其它正常组织中没有找到(Tedder et al.J.Immunol.135(2):973-979(1985))。CD20被认为调节细胞周期起始与分化的激活过程中的一个早期步骤(Tedder et al.,supra)，并且可能作为钙离子通道发挥作用(Tedder et al.J.Cell.Biochem.14D:195(1990))。CD20 antigen (also known as human B lymphocyte-restricted differentiation antigen, Bp35) is a hydrophobic transmembrane protein located on pre-B and mature B lymphocytes, with a molecular weight of approximately 35 kD (J. Biol. Chem. 264 (19) :11282-11287(1989); Einfeld et al. EMBO J.7(3):711-717(1988)). 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)).
本公开还提供了一种通过设计和表达包含靶向CD20的结构域与促三聚体结构域的重组蛋白，靶向结合T细胞表面的CD20，从而实现CD20聚集，诱导CD20的内吞，实现靶向T细胞定向标记、投递药物和核酸或实现检测或治疗的功能。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是仅在分化的成熟期B细胞表面上表达的135kDa B细胞限定的(B-cell-restricted)唾液酸糖蛋白(Dorken,B.等,J.Immunol.136:4470-4479(1986))。CD22在人中的最主要形式是CD22β，其含有胞外结构域中的7个免疫球蛋白超家族结构域(图1)(Wilson,G.L.等,J.Exp.Med.173:137-146(1991))。一种变体形式即CD22α缺乏免疫球蛋白超家族结构域3和4(Stamenkovic,I.和Seed,B.,Nature 345:74-77(1990))。已经显示了对人CD22的配体结合与免疫球蛋白超家族结构域1和2(也称为表位1和2)有关(Engel,P.等,J.Exp.Med.181:1581-1586(1995))。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 CD22β, 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, CD22α, lacks immunoglobulin superfamily domains 3 and 4 (Stamenkovic, I. and Seed, B., Nature 345:74-77 (1990)). Ligand binding to human CD22 has been shown to be associated with immunoglobulin superfamily domains 1 and 2 (also known as epitopes 1 and 2) (Engel, P. et al., J. Exp. Med. 181:1581-1586 (1995)).
本公开还提供了一种通过设计和表达包含靶向CD22的结构域与促三聚体结构域的重组蛋白，靶向结合T细胞表面的CD22，从而实现CD22聚集，诱导CD22的内吞，实现靶向T细胞定向标记、投递药物和核酸或实现检测或治疗的功能。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受体是在抗原呈递细胞表面发现的一种蛋白质，这些细胞包括B淋巴细胞、滤泡树突细胞、自然杀伤细胞、巨噬细胞、中性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞、人类血小板和肥大细胞，它们有助于保护免疫系统的功能。Fc受体的名字来源于它对被称为Fc(可结晶片段)区域的抗体的一部分的结合特异性。Fc受体与附着在受感染细胞或入侵病原体上的抗体结合。Fc受体可通过抗体介导的吞噬作用或抗体依赖性细胞介导的细胞毒效应，刺激吞噬细胞或细胞毒性细胞破坏微生物或感染细胞。一些病毒(如黄病毒)可通过抗体依赖性增强感染的机制增加病毒入侵细胞(Antibody-Dependent Enhancement,ADE)(Anderson R.Manipulation of cell surface macromolecules by flaviviruses.Adv Virus Res.2003；59:229-274.doi:10.1016/s0065-3527(03)59007-8)。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 (such as flaviviruses) 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).
本公开还提供了一种通过设计和表达包含靶向Fc受体的结构域与促三聚体结构域的重组蛋白，靶向结合T细胞表面的Fc受体，从而实现Fc受体聚集，诱导Fc受体的内吞，实现靶向T细胞定向标记、投递药物和核酸或实现检测或治疗的功能。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.
附图说明Description of the drawings
图1示出了融合蛋白的基本结构及其可能的蛋白结构形式，其中，图1A为融合蛋白的基本结构图，包括靶向结合结构域(Targeting Domain)，接头(Linker)，三聚体结构域(Trimer domain)；图1B为靶向TCR的融合蛋白(BMA-STII-VXT)的基本结构图，包括靶向TCR的单链抗体片段(ScFv)，Strep tag II标签，甘丝氨酸铰链区(Glycine-Serine linker)，胶原蛋白三聚体结构域(VXT)；图1C为靶向CD3的融合蛋白(BMC-STII-VXT)的基本结构图，包括靶向CD3的单链抗体片段(ScFv)，Strep tag II标签，甘丝氨酸铰链区(Glycine-Serine linker)，胶原蛋白三聚体结构域(VXT)；图1D为靶向CD5的融合蛋白(BMD-STII-VXT)的基本结构图，包括靶向CD5的单链抗体片段(ScFv)，Strep tag II标签，甘丝氨酸铰链区(Glycine-Serine linker)，胶原蛋白三聚体结构域(VXT)；图1E为靶向CD7的融合蛋白(BME-STII-VXT)的基本结构图，包括靶向CD7的单链抗体片段(ScFv)，Strep tag II标签，甘丝氨酸铰链区(Glycine-Serine linker)，胶原蛋白三聚体结构域(VXT)；图1F为靶向CD4的融合蛋白(BMF-STII-VXT)的基本结构图，包括靶向CD4的单链抗体片段(ScFv)，Strep tag II标签，甘丝氨酸铰链区(Glycine-Serine linker)，胶原蛋白三聚体结构域(VXT)；图1G为靶向CD20的融合蛋白(BMG-STII-VXT)的基本结构图，包括靶向CD20的单链抗体片段(ScFv)，Strep tag II标签，甘丝氨酸铰链区(Glycine-Serine linker)，胶原蛋白三聚体结构域(VXT)；图1H为靶向CD22的融合蛋白(BMH-STII-VXT)的基本结构图，包括靶向CD22的单链抗体片段(ScFv)，Strep tag II标签，甘丝氨酸铰链区(Glycine-Serine linker)，胶原蛋白三聚体结构域(VXT)；图1I为靶向CD64的融合蛋白(BMI-STII-VXT)的基本结构图，包括靶向CD64的单链抗体片段(ScFv)，Strep tag II标签，甘丝氨酸铰链区(Glycine-Serine linker)，胶原蛋白三聚体结构域(VXT)；图1J为融合蛋白可能的蛋白结构形式：单体，二聚体(靶向结构域形成异二聚体)，二聚体(靶向结构域形成同二聚体)，三聚体；图1K示出了靶向TCR的融合蛋白利用SDS-PAGE进行分离的结果，结果显示在还原状态，表达的蛋白是单体，在非还原状态，表达的蛋白是二聚体结构。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-VXT), including Single chain antibody fragment (ScFv) targeting CD5, Strep tag II tag, Glycine-Serine linker, collagen trimer domain (VXT); Figure 1E shows the fusion protein targeting CD7 (BME -STII-VXT) basic structural diagram, including single-chain antibody fragment (ScFv) targeting CD7, Strep tag II tag, Glycine-Serine linker, and collagen trimer domain (VXT); Figure 1F is a basic structural diagram of the CD4-targeting fusion protein (BMF-STII-VXT), including a CD4-targeting single-chain antibody fragment (ScFv), a Strep tag II tag, and a Glycine-Serine linker. Collagen 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 domain forms heterodimer), dimer (targeting domain forms homodimer), trimer; Figure 1K shows the targeting TCR The fusion protein was separated using SDS-PAGE. The results showed that in the reduced state, the expressed protein was a monomer, and in the non-reduced state, the expressed protein was a dimer structure.
图2示出了靶向TCR的融合蛋白(BMA-STII-VXT)进行Biotin修饰后对T细胞的特异性结合的流式检测结果，结果显示BMA-STII-VXT能够高效结合到T细胞表面，并能与TCR-αβ的抗体竞争性结合T细胞表面的 TCR。Figure 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. The results show that BMA-STII-VXT can efficiently bind to the surface of T cells. And can competitively bind to TCR-αβ antibodies on the surface of T cells TCR.
图3示出了靶向TCR的融合蛋白(BMA-STII-VXT)和TCR-ab抗体同时标记T细胞的流式细胞检测结果，图3A显示95.5％的T细胞都呈现TCR-αβ高表达，图3B显示BMA-STII-VXT能完全屏蔽TCR-αβ抗体，竞争结合T细胞；图3C显示BMA-STII-VXT能竞争性结合T细胞，使TCR-αβ的抗体不能结合T细胞；图3D显示结合TCR的BMA-VXT被内吞到T细胞内，部分TCR经过6个小时又重新回到T细胞表面。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-αβ. Figure 3B shows that BMA-STII-VXT can completely shield TCR-αβ antibodies and compete for binding to T cells; Figure 3C shows that BMA-STII-VXT can competitively bind to T cells, making TCR-αβ 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.
图4示出了靶向TCR的融合蛋白与内部包载核酸的LNP连接的结构示意图。Figure 4 shows a schematic structural diagram of the connection between a TCR-targeting fusion protein and an LNP encapsulating nucleic acid inside.
图5示出了靶向TCR的融合蛋白与内部包载转座子的LNP连接的结构示意图。Figure 5 shows a schematic structural diagram of the connection between a TCR-targeting fusion protein and an LNP containing a transposon internally.
图6示出了靶向TCR的融合蛋白与内部包载化疗药物的LNP连接的结构示意图。Figure 6 shows a schematic structural diagram of the connection between a TCR-targeting fusion protein and an LNP entrapping a chemotherapeutic drug internally.
具体实施方式Detailed ways
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。In the present invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used in this article are terms and routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.
如在本文中所使用的术语“约”指可测量的值，例如量、持续时间等，并且包括与指定值相差±20％、±10％、±5％、±1％、±0.5％或±0.1％的变化方式。The term "about" as used herein refers to a measurable value, such as an amount, duration, etc., and includes deviations from the specified value of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% variation.
如在本文中所使用的术语“抗体”指与抗原特异性结合的免疫球蛋白分子。抗体可以是源自天然来源或重组来源的完整免疫球蛋白并且可以是完整免疫球蛋白的免疫反应部分。抗体通常是免疫球蛋白分子的四聚体。本公开中的抗体可以以多种形式存在，包括例如多克隆抗体、单克隆抗体、纳米抗体、Fv、Fab和F(ab)，以及单链抗体、双链抗体、三链抗体、四链抗体和人源化抗体。The term "antibody" as used herein 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.
如本文所用，抗体的“抗体片段”或“抗原结合片段”指全长抗体的任何部分，其少于全长，但是至少包含结合抗原的所述抗体的部分可变区(例如一个或多个CDR和/或一个或多个抗体结合位点)，并且因此保留结合特异性以及所述全长抗体的至少部分特异性结合能力。因此，抗原结合片段指包含与衍生抗体片段的抗体结合相同抗原的抗原结合部分的抗体片段。抗体片段包括通过酶促处理全长抗体所产生的抗体衍生物，以及合成产生的衍生物，例如重组产生的衍生物。抗体包括抗体片段。抗体片段的实例包括但不限于Fab、Fab'、F(ab')2、单链Fv(scFv)、Fv、dsFv、双抗体、Fd和Fd'片段以及其他片段，包括修饰的片段(参见，例如，Methods in Molecular Biology，Vol 207:Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003)；Chapter 1；p 3-25,Kipriyanov)。所述片段可以包括连接在一起的多条链，例如通过二硫键和/或通过肽接头。抗体片段一般包含至少或约50个氨基酸，并且典型至少或约200个氨基酸。抗原结合片段包括任何抗体片段，其在被插入抗体框架(例如通过置换相应区域)时获得免疫特异性地结合(即表现出至少或至少约107-108M-1的Ka)抗原的抗体。“功能片段”或“抗CCR8抗体的类似物”是可防止或实质降低所述受体结合配体或启动信号转导的能力的片段或类似物。正如本文所使用，功能片段一般与“抗体片段”含义相同，且就抗体而论，可指能防止或实质降低所述受体结合配体或启动信号转导的能力的片段，例如Fv、Fab、F(ab')2等等。“Fv”片段由一条重链的可变结构域和一条轻链的可变结构域籍非共价结合方式而形成的二聚体(VH-VL二聚体)组成。在该构型中，每个可变结构域的三个CDRs相互作用，以确定VH-VL二聚体表面上的靶结合位点，与完整抗体的情况一样。所述六个CDRs共同赋予完整抗体的靶结合特异性。但是，即使是单个可变结构域(或仅包括3个靶特异的CDRs的Fv的一半)，仍可具有识别和结合靶的能力。就抗体链多肽序列而言，短语“基本相同”可理解为表现出与参照多肽序列至少60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更多的序列同一性的抗体链。就核酸序列而言，该术语可理解为表现出与参照核酸序列至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性的核苷酸序列。As used herein, 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. Thus, 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. Examples of 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. As used herein, 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. However, even a single variable domain (or half of an Fv including only 3 target-specific CDRs) may still have the ability to recognize and bind the target. With respect to an antibody chain polypeptide sequence, the 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. With respect to a 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.
序列“相同性”或“同一性”具有本领域公认的含义，并且可以利用公开的技术计算两个核酸或多肽分子或区域之间序列相同性的百分比。可以沿着多核苷酸或多肽的全长或者沿着该分子的区域测量序列相同性。虽然存在许多测量两个多核苷酸或多肽之间的相同性的方法，但是术语“相同性”是技术人员公知的(Carrillo,H.&Lipman,D.,SIAM J Applied Math 48:1073(1988))。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) ).
术语“膜受体蛋白”指细胞膜上或细胞内具有特定功能的蛋白质，能接受外界信号并将其转化为细胞内的一系列生化反应，引起细胞结构或功能的改变。有单体型受体和复合型受体之分。The term "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.
术语“载体”是包含分离的核酸并且可以用于将分离的核酸递送至细胞内部的物质组合物。许多载体是本领域已知的，包括但不限于线性多核苷酸、与离子或两性化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制质粒或病毒。该术语也应解释为包括有助于核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物，脂质体等。病毒载体的实例包括但不限于慢病毒、腺病毒载体、腺相关病毒载体、逆转录病毒载体等。非病毒载体的实例包括但不限于CRISPR载体系统、Sleeping Beauty转座子系统等。如在本文中所使用的“激活”指已经被充分刺激以诱导可检测的细胞增殖的T细胞的状态。激活还可以与诱导的细胞因子产生和可检测的效应子功能相关。术语“激活的T细胞”尤其指正在进行细胞分裂的T细胞。The term "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. Thus, the term "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. Examples of viral vectors include, but are not limited to, lentivirus, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and the like. Examples of non-viral vectors include, but are not limited to, CRISPR vector systems, Sleeping Beauty transposon systems, etc. "Activated" as used herein 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.
本公开提供一种融合蛋白，其包含与抗原结合的组件(module)和促进三聚体形成的组件，所述抗原包含能实现网格蛋白依赖性内吞机制(clathrin-dependent endocytosis，CDE)的细胞膜受体蛋白。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. Cell membrane receptor proteins.
在一些优选地实施方案中，所述细胞膜受体蛋白选自T细胞膜受体抗原、B细胞膜受体抗原和单核细胞等抗 原呈递细胞(Antigen present cell)膜受体抗原中的一种或多种。In some preferred embodiments, 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.
在一些优选地实施方案中，所述T细胞膜受体抗原选自TCR、CD3、CD4、CD5、CD7、CCR5、CXCR4中的一种或多种。In some preferred embodiments, the T cell membrane receptor antigen is selected from one or more of TCR, CD3, CD4, CD5, CD7, CCR5, and CXCR4.
在一些优选地实施方案中，所述B细胞膜受体抗原选自CD20和CD22。In some preferred embodiments, the B cell membrane receptor antigen is selected from CD20 and CD22.
在一些优选地实施方案中，所述单核细胞等抗原呈递细胞膜受体抗原选自Fc受体抗原。In some preferred embodiments, the monocyte and other antigen-presenting cell membrane receptor antigens are selected from Fc receptor antigens.
在一些优选地实施方案中，所述Fc受体抗原选自Fcα、FcγRI、FcγRII、FcγRIV、FcεRI和Fcα/μR中的一种或多种。In some preferred embodiments, the Fc receptor antigen is selected from one or more of Fcα, FcγRI, FcγRII, FcγRIV, FcεRI and Fcα/μR.
在一些优选地实施方案中，Fcα为FcαRI，更优选地为CD89。In some preferred embodiments, Fca is FcaRI, more preferably CD89.
在一些优选地实施方案中，FcγRI为CD64；优选地，FcγRII为FcγRIIB2(CD32)。In some preferred embodiments, FcyRII is CD64; preferably, FcyRII is FcyRIIB2 (CD32).
在一些优选地实施方案中，FcεRI为CD89。In some preferred embodiments, FcεRI is CD89.
在一些优选地实施方案中，前述促进三聚体形成的组件选自脂联素或其胶原样结构域、T4纤维蛋白三聚体结构域、人类胶原蛋白的三聚体结构，如：穿膜胶原蛋白XIII、XV、XVII和XVIII的氨基端NC1结构域，原纤维相关胶原蛋白IX、XII、XIV、XVI、XIX、XX和XXI的NC2结构域、GCN4三聚化结构域、人肺表面活性剂蛋白A(SP-A)、甘露糖结合蛋白A(MBP-A)、大肠杆菌天冬氨酸转氨甲酰酶(ATCase)的催化亚基、寡聚卷曲螺旋粘附素和包膜病毒I类融合蛋白的互补七肽重复区。In some preferred embodiments, 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.
在一些优选地实施方案中，所述促进三聚体形成的组件为XV的氨基端NC1结构域。In some preferred embodiments, the component that promotes trimer formation is the amino-terminal NC1 domain of XV.
在一些优选地实施方案中，所述促进三聚体形成的组件包含与SEQ ID NO.18所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列。In some preferred embodiments, 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.
在一些优选地实施方案中，所述促进三聚体形成的组件包含SEQ ID NO.18所示的氨基酸序列或者在SEQ ID NO:18上缺失、添加或取代1、2或3个残基所得到的氨基酸序列。In some preferred embodiments, 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.
在一些优选地实施方案中，前述与抗原结合的组件选自抗原的抗体或其抗原结合片段或与所述膜受体蛋白结合的配体。In some preferred embodiments, 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.
在一些优选地实施方案中，前述TCR抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.1或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.2或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.3或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO.4或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO.5或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.6或其任何变体的轻链CDR3。In some preferred embodiments, 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.
在一些优选地实施方案中，前述TCR抗原的抗体或其抗原结合片段包含选自下列的重链和轻链的CDR组合：In some preferred embodiments, 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:
分别包含SEQ ID NO:1、2、3的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO:4、5、6的轻链CDR1、CDR2及CDR3序列。Comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 1, 2 and 3 respectively, and/or the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 4, 5 and 6 respectively.
在一些优选地实施方案中，前述TCR抗体片段包含选自氨基酸序列SEQ ID NO.7或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO.8或其任何变体的轻链可变区。In some preferred embodiments, 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.
在一些优选地实施方案中，其中前述CD3抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.9或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.10或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.11或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO.12或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO.13或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.14或其任何变体的轻链CDR3。In some preferred embodiments, 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. A light chain CDR3 selected from the amino acid sequence SEQ ID NO. 14 or any variant thereof.
在一些优选地实施方案中，其中所述CD3抗体或其抗原结合片段分别包含氨基酸序列如SEQ ID NO:9、10、11所示的重链CDR1、CDR2及CDR3，和/或分别包含氨基酸序列如SEQ ID NO:12、13、14所示的轻链CDR1、CDR2及CDR3。In some preferred embodiments, 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.
在一些优选地实施方案中，其中所述CD3抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.15或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO.16或其任何变体的轻链可变区。In some preferred embodiments, wherein 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.
在一些优选地实施方案中，其中所述CD5抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.215或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.216或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.217或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO.218或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO.219或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.220或其任何变体的轻链CDR3。In some preferred embodiments, 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, A light chain CDR2 selected from the amino acid sequence SEQ ID NO. 219 or any variant thereof, a light chain CDR3 selected from the amino acid sequence SEQ ID NO. 220 or any variant thereof.
在一些优选地实施方案中，其中所述CD5抗原的抗体或其抗原结合片段分别包含氨基酸序列如SEQ ID NO:215、216、217所示的重链CDR1、CDR2及CDR3，和/或分别包含氨基酸序列如SEQ ID NO:218、219、220所示的轻链CDR1、CDR2及CDR3。In some preferred embodiments, 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.
在一些优选地实施方案中，其中所述CD5抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.221或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO.222或其任何变体的轻链可变区。In some preferred embodiments, 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.
在一些优选地实施方案中，其中所述CD7抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.223或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.224或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.225或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO.226或其任何变体的轻链CDR1，选自 氨基酸序列SEQ ID NO.227或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.228或其任何变体的轻链CDR3。In some preferred embodiments, 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.
在一些优选地实施方案中，其中所述CD7抗原的抗体或其抗原结合片段分别包含氨基酸序列如SEQ ID NO:223、224、225所示的重链CDR1、CDR2及CDR3，和/或分别包含氨基酸序列如SEQ ID NO:226、227、228所示的轻链CDR1、CDR2及CDR3。In some preferred embodiments, 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.
在一些优选地实施方案中，其中所述CD7抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.229或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO.230或其任何变体的轻链可变区。In some preferred embodiments, 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.
在一些优选地实施方案中，其中所述CD4抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.231或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.232或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.233或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO.234或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO.235或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.236或其任何变体的轻链CDR3。In some preferred embodiments, 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, A light chain CDR2 selected from the amino acid sequence SEQ ID NO. 235 or any variant thereof, a light chain CDR3 selected from the amino acid sequence SEQ ID NO. 236 or any variant thereof.
在一些优选地实施方案中，其中所述CD4抗原的抗体或其抗原结合片段分别包含氨基酸序列如SEQ ID NO:231、232、233所示的重链CDR1、CDR2及CDR3，和/或分别包含氨基酸序列如SEQ ID NO:234、235、236所示的轻链CDR1、CDR2及CDR3。In some preferred embodiments, 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.
在一些优选地实施方案中，其中所述CD4抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.237或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO.238或其任何变体的轻链可变区。In some preferred embodiments, 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.
在一些优选地实施方案中，其中所述CD20抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.239或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.240或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.241或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO.242或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO.243或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.244或其任何变体的轻链CDR3。In some preferred embodiments, 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.
在一些优选地实施方案中，其中所述CD20抗原的抗体或其抗原结合片段分别包含氨基酸序列如SEQ ID NO:239、240、241所示的重链CDR1、CDR2及CDR3，和/或分别包含氨基酸序列如SEQ ID NO:242、243、244所示的轻链CDR1、CDR2及CDR3。In some preferred embodiments, 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.
在一些优选地实施方案中，其中所述CD20抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.245或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO.246或其任何变体的轻链可变区。In some preferred embodiments, 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.
在一些优选地实施方案中，其中所述CD22抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.247或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.248或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.249或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO.250或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO.251或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.252或其任何变体的轻链CDR3。In some preferred embodiments, 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, A light chain CDR2 selected from the amino acid sequence SEQ ID NO. 251 or any variant thereof, a light chain CDR3 selected from the amino acid sequence SEQ ID NO. 252 or any variant thereof.
在一些优选地实施方案中，其中所述CD22抗原的抗体或其抗原结合片段分别包含氨基酸序列如SEQ ID NO:247、248、249所示的重链CDR1、CDR2及CDR3，和/或分别包含氨基酸序列如SEQ ID NO:250、251、252所示的轻链CDR1、CDR2及CDR3。In some preferred embodiments, 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.
在一些优选地实施方案中，其中所述CD22抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.253或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO.254或其任何变体的轻链可变区。In some preferred embodiments, 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.
在一些优选地实施方案中，其中所述CD64抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.255或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.256或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.257或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO.258或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO.259或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.260或其任何变体的轻链CDR3。In some preferred embodiments, 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.
在一些优选地实施方案中，其中所述CD64抗原的抗体或其抗原结合片段分别包含氨基酸序列如SEQ ID NO:255、256、257所示的重链CDR1、CDR2及CDR3，和/或分别包含氨基酸序列如SEQ ID NO:258、259、260所示的轻链CDR1、CDR2及CDR3。In some preferred embodiments, 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.
在一些优选地实施方案中，其中所述CD64抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.261或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO.262或其任何变体的轻链可变区。In some preferred embodiments, 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.
在一些优选地实施方案中，前述的融合蛋白中所述与抗原结合的组件和促进三聚体形成的组件可直接连接或通过接头L1连接。In some preferred embodiments, 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.
在一些优选地实施方案中，所述接头为柔性接头。In some preferred embodiments, the joint is a flexible joint.
在一些优选地实施方案中，所述融合蛋白还包含纯化标签；所述纯化标签在所述融合蛋白的N端、C端或在所述与抗原结合的组件和促进三聚体形成的组件之间。In some preferred embodiments, 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.
在一些优选地实施方案中，所述纯化标签在与抗原结合的组件和促进三聚体形成的组件之间。In some preferred embodiments, the purification tag is between a component that binds the antigen and a component that promotes trimer formation.
在一些优选地实施方案中，所述纯化标签选自GST标签、His标签、Myc标签、E标签、Strep标签和HA标签。 In some preferred embodiments, the purification tag is selected from the group consisting of GST tag, His tag, Myc tag, E tag, Strep tag and HA tag.
在一些优选地实施方案中，所述Strep标签为STII标签，其氨基酸序列如SEQ ID NO.17所示。In some preferred embodiments, the Strep tag is an STII tag, and its amino acid sequence is shown in SEQ ID NO. 17.
在一些优选地实施方案中，所述柔性接头L1选自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)、GGGGSGGGGSGGGGS(SEQ ID NO.24)、GGGGSGGGGSGGGG(SEQ ID NO.25)、GGGGSGGGGSGGGGSGGGGS(SEQ ID NO.26)、GGGGSGGGGSGGGSGGGGS(SEQ ID NO.27)、GSGSGSGS(SEQ ID NO.28)、GGSGSGSG(SEQ ID NO.29)、GGSGSG(SEQ ID NO.30)、GGSG(SEQ ID NO.31)。In some preferred embodiments, 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),GSGSGSGS(SEQ ID NO.27) NO.28), GGSGSGSG(SEQ ID NO.29), GGSGSG(SEQ ID NO.30), GGSG(SEQ ID NO.31).
在一些优选地实施方案中，所述接头L1选自GGGS(SEQ ID NO.19)、GGGGSGGGGS(SEQ ID NO.21)、GGGGSGGGGSGGGGSGGGGS(SEQ ID NO.26)和GGGGSGGGGSGGGSGGGGS(SEQ ID NO.27)。In some preferred embodiments, 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).
在一些优选地实施方案中，所述融合蛋白包含与SEQ ID NO:36所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；优选地，所述氨基酸序列为如SEQ ID NO:36所示的氨基酸序列或者在SEQ ID NO:36上缺失、添加或取代1、2或3个残基所得到的氨基酸序列。In some preferred embodiments, 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.
另一方面，本公开提供了编码前述融合蛋白的核酸。In another aspect, the present disclosure provides nucleic acids encoding the aforementioned fusion proteins.
在另一方面，本公开提供了包含前述核酸的载体。In another aspect, the present disclosure provides vectors comprising the aforementioned nucleic acids.
在另一方面，本公开提供了包含前述核酸或载体的细胞。In another aspect, the present disclosure provides cells comprising the aforementioned nucleic acids or vectors.
在另一方面，本公开提供了一种组合物，其包含前述融合蛋白、前述核酸、前述载体和/或前述的细胞。In another aspect, the present disclosure provides a composition comprising the aforementioned fusion protein, the aforementioned nucleic acid, the aforementioned vector and/or the aforementioned cell.
在另一方面，本公开提供一种前药，其包含选自下述任一：In another aspect, the present disclosure provides a prodrug comprising any of the following:
(1)前述融合蛋白和LNP；(1) The aforementioned fusion protein and LNP;
(2)前述融合蛋白与细胞毒素，其中所述融合蛋白和所述细胞毒素通过接头单元连接；或(2) The aforementioned fusion protein and cytotoxin, wherein the fusion protein and the cytotoxin are connected through a linker unit; or
(3)前述融合蛋白和标记。(3) The aforementioned fusion protein and tag.
在一个优选地实施方案中，其中所述融合蛋白和所述LNP通过接头L2连接；更优选地，所述接头L2为在柔性多肽的C末端加入了一个半胱氨酸残基的多肽；所述柔性多肽选自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)、GGGGSGGGGSGGGGS(SEQ ID NO.24)、GGGGSGGGGSGGGG(SEQ ID NO.25)、GGGGSGGGGSGGGGSGGGGS(SEQ ID NO.26)、GGGGSGGGGSGGGSGGGGS(SEQ ID NO.27)、GSGSGSGS(SEQ ID NO.28)、GGSGSGSG(SEQ ID NO.29)、GGSGSG(SEQ ID NO.30)、GGSG(SEQ ID NO.31)。In a preferred embodiment, 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. 23), GGGGSGGGGSGGGGS(SEQ ID NO.24),GGGGSGGGGSGGGG(SEQ ID NO.25),GGGGSGGGGSGGGGSGGGGS(SEQ ID NO.26),GGGGSGGGGSGGGSGGGGS(SEQ ID NO.27),GSGSGSGS(SEQ ID NO.28),GGSGSGSG(SEQ ID NO.26) .29), GGSGSG(SEQ ID NO.30), GGSG(SEQ ID NO.31).
在一个优选地实施方案中，所述接头L2选自GGGSC(SEQ ID NO.32)、GGGGSGGGGSC(SEQ ID NO.33)、GGGGSGGGGSGGGGSGGGGSC(SEQ ID NO.34)和GGGGSGGGGSGGGSGGGGSC(SEQ ID NO.35)。In a preferred embodiment, 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).
在一个实施方案中，所述LNP内包含至少一种具有生物活性的核酸、蛋白和小分子化合物；优选地，所述具有生物活性的核酸选自DNA和RNA中的一种或多种；优选地，所述小分子化合物为化疗药物；优选地，所述化疗药物为细胞毒素。In one embodiment, 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 Preferably, the small molecule compound is a chemotherapeutic drug; preferably, the chemotherapeutic drug is a cytotoxin.
在一个优选地实施方案中，具有生物活性的核酸选自编码嵌合抗原受体(CAR)的核酸、编码CNK复合体的核酸，编码促细胞凋亡蛋白的核酸和编码嵌合蛋白构建体的核酸中的一种或多种。In a preferred embodiment, 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. One or more nucleic acids.
在一个优选地实施方案中，所述具有生物活性的核酸为编码转座子的核酸，所述核酸包括目的基因的核酸和编码转座酶的核酸。In a preferred embodiment, the biologically active nucleic acid is a nucleic acid encoding a transposon, and the nucleic acid includes a nucleic acid of a target gene and a nucleic acid encoding a transposase.
在一个优选地实施方案中，所述目的基因选自前述嵌合蛋白受体(CAR)、嵌合自然杀伤细胞受体(CNK)复合体或嵌合蛋白构建体中的一种或多种，转入目的基因的具体过程参见图5(参见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)。In a preferred embodiment, 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, The specific process of transferring the target gene is shown in 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).
在一个优选地实施方案中，前述嵌合抗原受体(CAR)包含细胞外结构域、跨膜结构域、共刺激信号传导区域和胞内信号传导结构域，所述细胞外结构域结合的抗原选自：间皮素(MSLN)、B7-H3(CD276)、硫酸软骨素蛋白多糖4(CSPG4)、Muc 16、Claudin 18.2、Claudin 8、NY-ESO-1、CD19、CD22、CD23、骨髓增生性白血病蛋白MPL、CD30、CD32、CD20、CD70、CD99、CD123、CD138、CD179b、CD200R、CD324、Fc受体样5FcRH5、CD171、CS-1(信号传导淋巴细胞活化分子家族7SLAMF7)；所述跨膜结构域为CD8或CD28的跨膜区；所述共刺激分子选自MHC I类分子，TNF受体蛋白，免疫球蛋白样蛋白，细胞因子受体，整联蛋白，信号传导淋巴细胞活化分子(SLAM蛋白)，激活NK细胞受体，BTLA，Toll配体受体，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)；所述胞内信号传导结构域选自CD3ζ，CD3γ，CD3δ，CD3ε，CD5，CD22，CD79a，CD79b，CD278(“ICOS”)，FcεRI，CD66d，DAP10和DAP12。In a preferred embodiment, the aforementioned chimeric antigen receptor (CAR) 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, immunoglobulin-like proteins, cytokine receptors, integrins, and signaling lymphocyte activation molecules. (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 CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI, CD66d, DAP10 and DAP12.
在一个优选地实施方案中，本公开提供了一种多功能复合体(multi-functional complex)，其包含以下组件：In a preferred embodiment, the present disclosure provides a multi-functional complex comprising the following components:
(1)NK激活受体组件，其至少包含NK细胞激活受体或其功能变体，所述NK细胞激活受体包含：(a)NK细胞激活受体胞外结构域(ED)或其功能变体，(b)NK细胞激活受体跨膜结构域(TMD)或其功能变体，以及(c)NK细胞激活受体胞内结构域(ICD)或其功能变体；任选地，所述NK细胞激活受体胞外结构域或其功能变体、所述NK细胞激活受体跨膜结构域或其功能变体和/或所述NK细胞激活受体胞内结构域或其功能变体之间包 含铰链或接头；(1) 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;
(2)CNK信号转接组件，其至少包含(i)NK细胞信号转换器(adaptor)或其功能变体，所述NK细胞信号转换器包含：(a)NK细胞信号转换器胞外结构域(ED)或其功能变体，(b)NK细胞信号转换器跨膜结构域(TMD)或其功能变体，以及(c)NK细胞信号转换器胞内结构域(ICD)或其功能变体；任选地，所述NK细胞信号转换器胞外结构域或其功能变体、所述NK细胞信号转换器跨膜结构域或其功能变体和/或所述NK细胞信号转换器胞内结构域或其功能变体之间包含铰链或接头；和(2) 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 Optionally, the NK cell signal transducer extracellular domain or its functional variant, the NK cell signal transducer transmembrane domain or its functional variant and/or the NK cell signal transducer cell Contain hinges or linkers between intradomains or functional variants thereof; and
任选地，所述NK激活受体组件、所述CNK信号转接组件之间包含铰链或接头。Optionally, a hinge or a linker is included between the NK activating receptor component and the CNK signal transfer component.
在一些实施方案中，所述NK激活受体组件中的所述NK细胞激活受体选自NKG2D、NKG2C、NKG2E、NKG2F、NKG2H、CD94、KIR2DL4、KIR2DS1、KIR2DS2、KIR2DS4、KIR3DS1，天然细胞毒性受体、TRAIL、DNAM-1、CD16a、2B4、NTB-A、CRACC以及NKp80；优选地，所述天然细胞毒性受体选自NKp46、NKp44和NKp30。In some embodiments, 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.
在一些优选的实施方案中，所述NK细胞激活受体是哺乳动物来源的NK细胞激活受体；优选地，所述哺乳动物选自人、灵长类动物、鼠、马、牛、绵羊、山羊、猫、猪、狗、美洲驼、羊驼、大象、松鼠、豚鼠。In some preferred embodiments, 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.
在一些优选的实施方案中，所述NK细胞激活受体是包含不同来源NK细胞激活受体结构域的重组NK细胞激活受体。In some preferred embodiments, the NK cell activating receptor is a recombinant NK cell activating receptor comprising NK cell activating receptor domains from different sources.
在一些优选的实施方案中，所述NK细胞激活受体是人源的NK细胞激活受体；优选地，所述NK细胞激活受体是包含不同的人源NK细胞激活受体结构域的重组NK细胞激活受体。In some preferred embodiments, the NK cell activating receptor is a human NK cell activating receptor; preferably, the NK cell activating receptor is a recombinant comprising different human NK cell activating receptor domains. NK cell activating receptor.
在一些优选的实施方案中，所述NK细胞激活受体是鼠源的NK细胞激活受体；优选地，所述NK细胞激活受体是包含不同的鼠源NK细胞激活受体结构域的重组NK细胞激活受体。In some preferred embodiments, the NK cell activating receptor is a murine NK cell activating receptor; preferably, the NK cell activating receptor is a recombinant comprising different murine NK cell activating receptor domains. NK cell activating receptor.
在一些优选的实施方案中，所述NK细胞激活受体是包含人源和鼠源NK细胞激活受体结构域的重组NK细胞激活受体。In some preferred embodiments, the NK cell activating receptor is a recombinant NK cell activating receptor comprising human and murine NK cell activating receptor domains.
在一些优选的实施方案中，所述NK细胞激活受体的胞外结构域为人或鼠的NK细胞激活受体的胞外结构域。In some preferred embodiments, the extracellular domain of the NK cell activating receptor is the extracellular domain of a human or murine NK cell activating receptor.
在一些优选的实施方案中，所述NK细胞激活受体的跨膜结构域为人或鼠的NK细胞激活受体的跨膜结构域。In some preferred embodiments, the transmembrane domain of the NK cell activating receptor is that of a human or murine NK cell activating receptor.
在一些优选的实施方案中，所述NK细胞激活受体的胞内结构域为人或鼠的NK细胞激活受体的胞内结构域。In some preferred embodiments, the intracellular domain of the NK cell activating receptor is the intracellular domain of a human or murine NK cell activating receptor.
在一些优选的实施方案中，所述NK细胞激活受体功能变体选自NK细胞激活受体的突变体，野生型融合蛋白，或野生型与突变型的融合蛋白。在一些优选的实施方案中，人NKG2D的胞外域包含与SEQ ID NO.37所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2D的胞外域的氨基酸序列如SEQ ID NO.37所示。In some preferred embodiments, the functional variant of the NK cell activating receptor is selected from a mutant of the NK cell activating receptor, a wild-type fusion protein, or a fusion protein of a wild-type and a mutant type. In some preferred embodiments, the extracellular domain of human NKG2D comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人NKG2D的全长序列包含与SEQ ID NO.38所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2D的全长序列的氨基酸序列如SEQ ID NO.38所示。In some preferred embodiments, 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.
在一些优选的实施方案中，小鼠NKG2D的胞外域包含与SEQ ID NO.39所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；小鼠NKG2D的胞外域的氨基酸序列如SEQ ID NO.39所示。In some preferred embodiments, the extracellular domain of mouse NKG2D comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，小鼠NKG2D的全长序列包含与SEQ ID NO:40所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；小鼠NKG2D的全长序的氨基酸序列如SEQ ID NO.40所示。In some preferred embodiments, the full-length sequence of mouse NKG2D comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 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.
在一些优选的实施方案中，人鼠重组NKG2D的全长序列包含与SEQ ID NO:41所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人鼠重组NKG2D的全长序列的氨基酸序列如SEQ ID NO.41所示。In some preferred embodiments, the full-length sequence of human mouse recombinant NKG2D comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 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.
在一些优选的实施方案中，人NKG2C的全长序列包含与SEQ ID NO.42所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2C的全长序列的氨基酸序列如SEQ ID NO.42所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人NKG2E的全长序列包含与SEQ ID NO：43所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2E的全长序列的氨基酸序列如SEQ ID NO.43所示。In some preferred embodiments, the full-length sequence of human NKG2E comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 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.
在一些优选的实施方案中，人NKG2F的全长序列包含与SEQ ID NO：44所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2F的全长序列的氨基酸序列如SEQ ID NO.44所示。In some preferred embodiments, the full-length sequence of human NKG2F comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 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.
在一些优选的实施方案中，人CD94的全长序列包含与SEQ ID NO.45所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人CD94的氨基酸序列如SEQ ID NO.45所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人KIR2DL4的全长序列包含与SEQ ID NO：46所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具 有98％或99％以上同一性的氨基酸序列；人KIR2DL4的全长序列的氨基酸序列如SEQ ID NO.46所示。In some preferred embodiments, the full-length sequence of human KIR2DL4 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 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.
在一些优选的实施方案中，人KIR2DS1的全长序列包含与SEQ ID NO：47所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR2DS1的全长序列的氨基酸序列如SEQ ID NO.47所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人KIR2DS2的全长序列包含与SEQ ID NO：48所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR2DS2的全长序列的氨基酸序列如SEQ ID NO.48所示。In some preferred embodiments, the full-length sequence of human KIR2DS2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 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.
在一些优选的实施方案中，人KIR2DS4的全长序列包含与SEQ ID NO：49所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR2DS4的全长序列的氨基酸序列如SEQ ID NO.49所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人KIR3DS1的全长序列包含与SEQ ID NO：50所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR3DS1的全长序列的氨基酸序列如SEQ ID NO.50所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人NKp46的全长序列包含与SEQ ID NO.51所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKp46的全长序列的氨基酸序列如SEQ ID NO.51所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人NKp44的全长序列包含与SEQ ID NO.52所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKp44的全长序列的氨基酸序列如SEQ ID NO.52所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人NKp30的全长序列包含与SEQ ID NO.53所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKp30的全长序列的氨基酸序列如SEQ ID NO.53所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人DNAM1的全长序列包含与SEQ ID NO：54所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DNAM1的全长序列的氨基酸序列如SEQ ID NO.54所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人TRAIL的全长序列包含与SEQ ID NO：55所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人TRAIL的全长序列的氨基酸序列如SEQ ID NO.55所示。In some preferred embodiments, the full-length sequence of human TRAIL comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 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.
在一些优选的实施方案中，人CD16a的全长序列包含与SEQ ID NO：56所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人CD16a的全长序列的氨基酸序列如SEQ ID NO.56所示。In some preferred embodiments, the full-length sequence of human CD16a comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 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.
在一些优选的实施方案中，人2B4的全长序列包含与SEQ ID NO：57所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人2B4的全长序列的氨基酸序列如SEQ ID NO.57所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人NTB-A的全长序列包含与SEQ ID NO：58所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NTB-A的全长序列的氨基酸序列如SEQ ID NO.58所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人CRACC的全长序列包含与SEQ ID NO：59所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人CRACC的全长序列的氨基酸序列如SEQ ID NO.59所示。In some preferred embodiments, the full-length sequence of human CRACC comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 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.
在一些优选的实施方案中，人NKp80的全长序列包含与SEQ ID NO：60所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKp80的全长序列的氨基酸序列如SEQ ID NO.60所示。In some preferred embodiments, 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.
在一些实施方案中，所述CNK信号转接组件中的所述NK细胞信号转换器为DAP10或DAP12。In some embodiments, the NK cell signal transducer in the CNK signal transduction component is DAP10 or DAP12.
在一些优选的实施方案中，所述NK细胞信号转换器是哺乳动物来源的NK细胞信号转换器；优选地，所述哺乳动物选自人、灵长类动物、鼠、马、牛、绵羊、山羊、猫、猪、狗、美洲驼、羊驼、大象、松鼠、豚鼠。In some preferred embodiments, 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.
在一些优选的实施方案中，所述NK细胞信号转换器是包含不同来源NK细胞信号转换器结构域的重组NK细胞信号转换器。In some preferred embodiments, the NK cell signal transducer is a recombinant NK cell signal transducer comprising NK cell signal transducer domains from different sources.
在一些优选的实施方案中，所述NK细胞信号转换器是人源的NK细胞信号转换器；优选地，所述NK细胞信号转换器是包含不同的人源NK细胞信号转换器结构域的重组NK细胞信号转换器。In some preferred embodiments, the NK cell signal transducer is a human NK cell signal transducer; preferably, the NK cell signal transducer is a recombinant comprising different human NK cell signal transducer domains. NK cell signal transducer.
在一些优选的实施方案中，所述NK细胞信号转换器是鼠源的NK细胞信号转换器；优选地，所述NK细胞信号转换器是包含不同的鼠源NK细胞信号转换器结构域的重组NK细胞信号转换器。In some preferred embodiments, the NK cell signal transducer is a murine NK cell signal transducer; preferably, the NK cell signal transducer is a recombinant comprising different murine NK cell signal transducer domains. NK cell signal transducer.
在一些优选的实施方案中，所述NK细胞信号转换器是包含人源和鼠源NK细胞信号转换器结构域的重组NK细胞信号转换器。In some preferred embodiments, the NK cell signal transducer is a recombinant NK cell signal transducer comprising human and murine NK cell signal transducer domains.
在一些优选的实施方案中，所述NK细胞信号转换器的胞外结构域为人或鼠的NK细胞信号转换器的胞外结构 域。In some preferred embodiments, the extracellular domain of the NK cell signal transducer is an extracellular structure of a human or murine NK cell signal transducer. area.
在一些优选的实施方案中，所述NK细胞信号转换器的跨膜结构域为人或鼠的NK细胞信号转换器的跨膜结构域。In some preferred embodiments, the transmembrane domain of the NK cell signal transducer is a transmembrane domain of a human or murine NK cell signal transducer.
在一些优选的实施方案中，所述NK细胞信号转换器的胞内结构域为人或鼠的NK细胞信号转换器的胞内结构域；In some preferred embodiments, the intracellular domain of the NK cell signal transducer is an intracellular domain of a human or murine NK cell signal transducer;
在一些优选的实施方案中，所述CNK细胞信号转换器功能变体选自DAP10突变体或DAP12的突变体，或DAP10和DAP12的融合蛋白，或野生型DAP10或DAP12与突变型DAP10或DAP12的融合蛋白。In some preferred embodiments, 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.
在一些优选的实施方案中，所述CNK信号转接组件还包含(ii)免疫受体活化信号传导结构域(ITAM)和/或(iii)T细胞共刺激信号传导结构域。In some preferred embodiments, the CNK signaling component further comprises (ii) an immunoreceptor activation signaling domain (ITAM) and/or (iii) a T cell costimulatory signaling domain.
在一些优选的实施方案中，所述NK细胞信号转换器或其功能变体、所述免疫受体活化信号传导结构域(ITAM)和/或所述T细胞共刺激信号传导结构域之间包含铰链或接头；优选地，所述NK细胞信号转换器或其功能变体与所述免疫受体活化信号传导结构域(ITAM)域融合。In some preferred embodiments, 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; Preferably, the NK cell signal transducer or functional variant thereof is fused to the immune receptor activation signaling domain (ITAM) domain.
在一些优选的实施方案中，所述免疫受体活化信号传导结构域(ITAM)来自免疫受体的胞内激活信号传导结构域；优选地，所免疫受体选自TCRζ、CD2、CD3γ、CD3δ、CD3ε、CD3ζ、CD5、CD22、FcRγ、CD66d、FcαRI、FcγRI、FcγRII、FcγRIII、Dectin-1、CLEC-1、CD72、CD79A、CD79B；优选地，所述免疫受体活化信号传导结构域(ITAM)与NK细胞信号转换器或其功能变体融合；优选地，所述免疫受体是CD3ζ。In some preferred embodiments, the immune receptor activation signaling domain (ITAM) is from an intracellular activation signaling domain of an immune receptor; preferably, the immune receptor is selected from TCRζ, CD2, CD3γ, CD3δ , CD3ε, CD3ζ, CD5, CD22, FcRγ, CD66d, FcαRI, FcγRI, FcγRII, FcγRIII, Dectin-1, CLEC-1, CD72, CD79A, CD79B; Preferably, the immune receptor activation signaling domain (ITAM ) is fused to an NK cell signal transducer or a functional variant thereof; preferably, the immune receptor is CD3ζ.
在一些优选的实施方案中，所述T细胞共刺激信号传导结构域来自共刺激分子的胞内信号结构域；优选地，所述共刺激分子选自MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白质、细胞因子受体、整联蛋白、淋巴细胞活化信号分子(SLAM蛋白)、活化NK细胞受体、BTLA、Toll配体受体、OX40、CD2、CD7、CD16、CD27、CD28、CD30、CD40、CD38、CD35、CD79A、CD79B、CDS、ICAM-1、LFA-1、(CD11a/CD18)、4-1BB(CD137)、B7-H3、CDS、ICAM-1、ICOS(CD278)、GITR、BAFFR、LIGHT、HVEM(LIGHTR)、KIRDS2、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、NKG2D、NKG2C、NCR、DAP10、DAP12、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD19a、与CD83特异性结合的配体、CARD11、FcRa、FcRp、FcRy、Fyn、HVEM、ICOS、Lck、LAG3、LAT、LRP、NOTCH1、Wnt、OX40、ROR2、Ryk、SLAMF1、Slp76、pTa、TCRa、TCRp、TRIM、ZAP70、PTCH2。In some preferred embodiments, 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, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, NCR, DAP10, DAP12, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100SEMA4D ), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, Ligands that specifically bind to CD83, CARD11, FcRa, FcRp, FcRy, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, Wnt, OX40, ROR2, Ryk, SLAMF1, Slp76, pTa, TCRa, TCRp , TRIM, ZAP70, PTCH2.
在一些优选的实施方案中，人DAP10的全长序列包含与SEQ ID NO.61所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10的全长序列的氨基酸序列如SEQ ID NO.61所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人DAP10的全长序列包含与SEQ ID NO.62所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10的全长序列的氨基酸序列如SEQ ID NO.62所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人DAP10的跨膜结构域包含与SEQ ID NO.63所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10的跨膜结构域的氨基酸序列如SEQ ID NO.63所示。In some preferred embodiments, the transmembrane domain of human DAP10 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP12的全长序列包含与SEQ ID NO.64所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12的全长序列的氨基酸序列如SEQ ID NO.64所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人DAP12的跨膜结构域包含与SEQ ID NO.65所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12的跨膜结构域的氨基酸序列如SEQ ID NO.65所示。In some preferred embodiments, the transmembrane domain of human DAP12 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP10和人DAP12的跨膜结构域融合蛋白包含与SEQ ID NO.66所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10和人DAP12的跨膜结构域的氨基酸序列如SEQ ID NO.66所示。In some preferred embodiments, the transmembrane domain fusion protein of human DAP10 and human DAP12 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP10-DAP12融合蛋白序列包含与SEQ ID NO.67所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-DAP12融合蛋白序列的氨基酸序列如SEQ ID NO.67所示。In some preferred embodiments, the human DAP10-DAP12 fusion protein sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人CD3zeta胞内信号传导结构域序列包含与SEQ ID NO.68所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序 列，更优选具有98％或99％以上同一性的氨基酸序列；人CD3zeta胞内信号传导结构域序列的氨基酸序列如SEQ ID NO.68所示。In some preferred embodiments, the human CD3zeta intracellular signaling domain sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP10-CD3zeta序列包含与SEQ ID NO.69所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-CD3zeta序列的氨基酸序列如SEQ ID NO.69所示。In some preferred embodiments, the human DAP10-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP12-CD3zeta序列包含与SEQ ID NO.70所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-CD3zeta序列的氨基酸序列如SEQ ID NO.70所示。In some preferred embodiments, the human DAP12-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP10-DAP12-CD3zeta序列包含与SEQ ID NO.71所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-DAP12-CD3zeta序列的氨基酸序列如SEQ ID NO.71所示。In some preferred embodiments, the human DAP10-DAP12-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人41BB胞内信号传导结构域序列包含与SEQ ID NO.72所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人41BB胞内信号传导结构域序列的氨基酸序列如SEQ ID NO.72所示。In some preferred embodiments, the human 41BB intracellular signaling domain sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP10-41BB序列包含与SEQ ID NO.73所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-41BB序列的氨基酸序列如SEQ ID NO.73所示。In some preferred embodiments, the human DAP10-41BB sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP10-41BB-CD3zeta序列包含与SEQ ID NO.74所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-41BB-CD3zeta序列的氨基酸序列如SEQ ID NO.74所示。In some preferred embodiments, the human DAP10-41BB-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人CD28胞内信号传导结构域序列包含与SEQ ID NO.75所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人CD28胞内信号传导结构域序列的氨基酸序列如SEQ ID NO.75所示。In some preferred embodiments, the human CD28 intracellular signaling domain sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP10-CD28序列包含与SEQ ID NO.76所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-CD28序列的氨基酸序列如SEQ ID NO.76所示。In some preferred embodiments, the human DAP10-CD28 sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP10-CD28-CD3zeta序列包含与SEQ ID NO.77所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-CD28-CD3zeta序列的氨基酸序列如SEQ ID NO.77所示。In some preferred embodiments, the human DAP10-CD28-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP12-41BB序列包含与SEQ ID NO.78所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-41BB序列的氨基酸序列如SEQ ID NO.78所示。In some preferred embodiments, the human DAP12-41BB sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP12-41BB-CD3zeta序列包含与SEQ ID NO.79所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-41BB-CD3zeta序列的氨基酸序列如SEQ ID NO.79所示。In some preferred embodiments, the human DAP12-41BB-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP12-CD28序列包含与SEQ ID NO.80所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-CD28序列的氨基酸序列如SEQ ID NO.80所示。In some preferred embodiments, the human DAP12-CD28 sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP12-CD28-CD3zeta序列包含与SEQ ID NO.81所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-CD28-CD3zeta序列的氨基酸序列如SEQ ID NO.81所示。In some preferred embodiments, the human DAP12-CD28-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些实施方案中，在所述UT组件的靶向降解TCR的重组蛋白分子中，所述靶向TCR的结合蛋白分子结构域或其功能变体来自TCR抗体或其功能片段或其组合。In some embodiments, in the recombinant protein molecule of the UT component that targets TCR degradation, 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.
在一些优选的实施方案中，所述抗体选自TCRα抗体、TCRβ抗体、TCRαβ抗体、TCRγ抗体、TCRδ抗体、TCRγδ抗体、TCR Vδ2抗体、TCR Cβ1抗体；所述抗体的功能片段选自Fd、Fv、Fab、Fab’、F(ab’)2、Fv(scFv)、单链抗体(scFv)或纳米抗体(nanobody)、双链抗体、三链抗体和四链抗体；优选地，所述TCR抗体是TCR单链抗体。In some preferred embodiments, the antibody is selected from TCRα antibody, TCRβ antibody, TCRαβ antibody, TCRγ antibody, TCRδ antibody, TCRγδ antibody, TCR Vδ2 antibody, TCR Cβ1 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.
在一些优选的实施方案中，所述UT组件中的ERAD降解结构域来自HCMV糖蛋白US2、US3、US11或 US10、腺病毒E3-19K或HHV-7US21。In some preferred embodiments, the ERAD degradation domain in the UT component is from HCMV glycoprotein US2, US3, US11 or US10, adenovirus E3-19K or HHV-7US21.
在一些优选的实施方案中，所述HCMV糖蛋白US2的全长序列包含与SEQ ID NO.82所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US2的全长序列的氨基酸序列如SEQ ID NO.82所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HCMV糖蛋白US2的HLA结合结构域包含与SEQ ID NO.83所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US2的HLA结合结构域的氨基酸序列如SEQ ID NO.83所示。In some preferred embodiments, the HLA binding domain of the HCMV glycoprotein US2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，所述HCMV糖蛋白US2的ERAD降解结构域包含与SEQ ID NO.84所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US2的ERAD降解结构域的氨基酸序列如SEQ ID NO.84所示。In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，所述HCMV糖蛋白US3的全长序列包含与SEQ ID NO.85所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US3的全长序列的氨基酸序列如SEQ ID NO.85所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HCMV糖蛋白US3的HLA结合结构域包含与SEQ ID NO.86所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US3的HLA结合结构域的氨基酸序列如SEQ ID NO.86所示。In some preferred embodiments, the HLA binding domain of the HCMV glycoprotein US3 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，所述HCMV糖蛋白US3的ERAD降解结构域包含与SEQ ID NO.87所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US3的ERAD降解结构域的氨基酸序列如SEQ ID NO.87所示。In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US3 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，所述HCMV糖蛋白US11的全长序列包含与SEQ ID NO.88所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US11的全长序列的氨基酸序列如SEQ ID NO.88所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HCMV糖蛋白US11的MHC结合结构域包含与SEQ ID NO.89所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US11的MHC结合结构域的氨基酸序列如SEQ ID NO.89所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HCMV糖蛋白US11的ERAD降解结构域包含与SEQ ID NO.90所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US11的ERAD降解结构域的氨基酸序列如SEQ ID NO.90所示。In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US11 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，所述HCMV糖蛋白US10的全长序列包含与SEQ ID NO.91所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US10的全长序列的氨基酸序列如SEQ ID NO.91所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HCMV糖蛋白US10的HLA结合结构域包含与SEQ ID NO.92所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US10的HLA结合结构域的氨基酸序列如SEQ ID NO.92所示。In some preferred embodiments, the HLA binding domain of the HCMV glycoprotein US10 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，所述HCMV糖蛋白US10的ERAD降解结构域包含与SEQ ID NO.93所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV糖蛋白US10的ERAD降解结构域的氨基酸序列如SEQ ID NO.93所示。In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US10 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，所述腺病毒E3-19K的全长序列包含与SEQ ID NO.94所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述腺病毒E3-19K的全长序列的氨基酸序列如SEQ ID NO.94所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述腺病毒E3-19K的MHC结合结构域包含与SEQ ID NO.95所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序 列，更优选具有98％或99％以上同一性的氨基酸序列；所述腺病毒E3-19K的MHC结合结构域的氨基酸序列如SEQ ID NO.95所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述腺病毒E3-19K的ERAD降解结构域包含与SEQ ID NO.96所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述腺病毒E3-19K的ERAD降解结构域的氨基酸序列如SEQ ID NO.96所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HHV-7US21的全长序列包含与SEQ ID NO.97所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HHV-7US21的全长序列的氨基酸序列如SEQ ID NO.97所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HHV-7US21的MHC结合结构域包含与SEQ ID NO.98所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HHV-7US21的MHC结合结构域的氨基酸序列如SEQ ID NO.98所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HHV-7US21的ERAD降解结构域包含与SEQ ID NO.99所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HHV-7US21的ERAD降解结构域的氨基酸序列如SEQ ID NO.99所示。In some preferred embodiments, 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.
在一些实施方案中，所述UT组件还包含(ii)靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体。In some embodiments, the UT component further comprises (ii) a binding protein molecular domain or functional variant thereof that targets MHC I and/or MHC II.
在一些优选的实施方案中，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体为靶向HLA的结合蛋白分子结构域或其功能变体。In some preferred embodiments, 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.
在一些优选的实施方案中，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体来自抑制MHC分子表达或促进其降解的病毒内质网蛋白；优选地，所述病毒内质网糖蛋白选自HCMV US6、HSV ICP47、CPXV012、HPV E6/E7、EBV BNFL2a或BHV UL49.5；优选地，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体包含TAP结合结构域。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HCMV US6的全长序列包含与SEQ ID NO.100所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV US6的全长序列的氨基酸序列如SEQ ID NO.100所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HHV-7US6的TAP结合结构域包含与SEQ ID NO.101所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HHV-7US6的TAP结合结构域的氨基酸序列如SEQ ID NO.101所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HSV ICP47的全长序列包含与SEQ ID NO.102所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HSV ICP47的全长序列的氨基酸序列如SEQ ID NO.102所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HSV ICP47的TAP结合结构域包含与SEQ ID NO.103所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HSV ICP47的TAP结合结构域的氨基酸序列如SEQ ID NO.103所示。In some preferred embodiments, the TAP binding domain of HSV ICP47 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，所述CPXV012的全长序列包含与SEQ ID NO.104所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述CPXV012的全长序列的氨基酸序列如SEQ ID NO.104所示。In some preferred embodiments, the full-length sequence of CPXV012 includes an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，所述CPXV012的TAP结合结构域包含与SEQ ID NO.105所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述CPXV012的TAP结合结构域的氨基酸序列如SEQ ID NO.105所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述EBV BNFL2a的全长序列包含与SEQ ID NO.106所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述EBV BNFL2a的全长序列的氨基酸序列如SEQ ID NO.106所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述EBV BNFL2a的TAP结合结构域包含与SEQ ID NO.107所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述EBV BNFL2a的TAP结合结构域的氨基酸序列如SEQ  ID NO.107所示。In some preferred embodiments, the TAP binding domain of EBV BNFL2a comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，所述BHV UL49.5的全长序列包含与SEQ ID NO.108所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述BHV UL49.5的全长序列的氨基酸序列如SEQ ID NO.108所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述BHV UL49.5的TAP结合结构域包含与SEQ ID NO.109所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述BHV UL49.5的TAP结合结构域的氨基酸序列如SEQ ID NO.113所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体来自降解MHC和/或MHC II分子的病毒糖蛋白；优选地，病毒糖蛋白选自HCMV糖蛋白US2、US3、US11或US10、腺病毒E3-19K或HHV-7US21。In some preferred embodiments, 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.
在一些优选的实施方案中，所述US2的全长序列包含与SEQ ID NO.110所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US2的全长序列的氨基酸序列如SEQ ID NO.110所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述US2的HLA结合结构域包含与SEQ ID NO.111所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US2的HLA结合结构域的氨基酸序列如SEQ ID NO.111所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述US2的ERAD降解结构域包含与SEQ ID NO.112所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US2的ERAD降解结构域的氨基酸序列如SEQ ID NO.112所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述US3的全长序列包含与SEQ ID NO.113所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US3的全长序列的氨基酸序列如SEQ ID NO.113所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述US3的HLA结合结构域包含与SEQ ID NO.114所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US3的HLA结合结构域的氨基酸序列如SEQ ID NO.114所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述US3的ERAD降解结构域包含与SEQ ID NO.115所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US3的ERAD降解结构域的氨基酸序列如SEQ ID NO.115所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述US11的全长序列包含与SEQ ID NO.116所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US11的全长序列的氨基酸序列如SEQ ID NO.116所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述US11的HLA结合结构域包含与SEQ ID NO.117所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US11的HLA结合结构域的氨基酸序列如SEQ ID NO.117所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述US11的ERAD降解结构域包含与SEQ ID NO.118所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述US11的ERAD降解结构域的氨基酸序列如SEQ ID NO.118所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体还包含定向抑制或降解MICA、MICB、ULBP1、ULBP2、ULBP3、ULBP4、ULBP5或ULBP6的NK靶点蛋白的病毒蛋白；优选地，所述病毒蛋白选自HCMV UL16、UL141、UL142或腺病毒E3-19K。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HCMV UL16的全长序列包含与SEQ ID NO.119所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL16的全长序列的氨基酸序列如SEQ ID NO.119所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HCMV UL16的NK靶点蛋白结合结构域包含与SEQ ID NO.120所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL16的NK靶点蛋白结合结构域的氨 基酸序列如SEQ ID NO.120所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HCMV UL16的ERAD降解结构域包含与SEQ ID NO.121所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL16的ERAD降解结构域的氨基酸序列如SEQ ID NO.121所示。In some preferred embodiments, the ERAD degradation domain of HCMV UL16 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，所述HCMV UL141的全长序列包含与SEQ ID NO.122所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL141的全长序列的氨基酸序列如SEQ ID NO.122所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HCMV UL141的NK靶点蛋白结合结构域包含与SEQ ID NO.123所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL141的NK靶点蛋白结合结构域的氨基酸序列如SEQ ID NO.123所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HCMV UL141的ERAD降解结构域包含与SEQ ID NO.124所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL141的ERAD降解结构域的氨基酸序列如SEQ ID NO.124所示。In some preferred embodiments, the ERAD degradation domain of HCMV UL141 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，所述HCMV UL142的全长序列包含与SEQ ID NO.125所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL142的全长序列的氨基酸序列如SEQ ID NO.125所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HCMV UL142的MICA、ULBP3结合结构域包含与SEQ ID NO.126所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL142的MICA、ULBP3结合结构域的氨基酸序列如SEQ ID NO.126所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HCMV UL142的高尔基驻留结构域包含与SEQ ID NO.127所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HCMV UL142的高尔基驻留结构域的氨基酸序列如SEQ ID NO.127所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体还包含把MHC I分子从高尔基体转运到溶酶体进行降解的病毒蛋白；优选地，所述病毒蛋白选自HIV Nef、HIV Vpu、HHV-7U21、HHV-8KK3、HHV-8KK5、MHV-68MK3和HTLV-1p12。In some preferred embodiments, 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; Preferably, the viral protein is selected from HIV Nef, HIV Vpu, HHV-7U21, HHV-8KK3, HHV-8KK5, MHV-68MK3 and HTLV-1p12.
在一些优选的实施方案中，所述HIV Nef包含与SEQ ID NO.128所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HIV Nef的氨基酸序列如SEQ ID NO.128所示。In some preferred embodiments, the HIV Nef comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，所述HIV Vpu包含与SEQ ID NO.129所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HIV Vpu的氨基酸序列如SEQ ID NO.129所示。In some preferred embodiments, the HIV Vpu comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，所述HHV-8KK3包含与SEQ ID NO.130所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HHV-8KK3的氨基酸序列如SEQ ID NO.130所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HHV-8KK5包含与SEQ ID NO.131所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HHV-8KK5的氨基酸序列如SEQ ID NO.131所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述MHV-68MK3包含与SEQ ID NO.132所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述MHV-68MK3的氨基酸序列如SEQ ID NO.132所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HTLV-1p12包含与SEQ ID NO.133所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述HTLV-1p12的氨基酸序列如SEQ ID NO.133所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述靶向MHC I和/或MHC II的结合蛋白分子结构域或其功能变体还包含介导MHC-多肽分子从高尔基体返回内质网并促进其降解的病毒蛋白；优选地，所述病毒蛋白包含MHC结合结构和KDEL receptor结合结构域；优选地，所述病毒蛋白是Cowpox Virus蛋白CPXV203。In some preferred embodiments, 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.
在一些优选的实施方案中，所述牛痘病毒蛋白CPXV203的全长序列包含与SEQ ID NO.134所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸 序列，更优选具有98％或99％以上同一性的氨基酸序列；所述牛痘病毒蛋白CPXV203的全长序列的氨基酸序列如SEQ ID NO.134所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述牛痘病毒蛋白CPXV203的MHC结合结构域包含与SEQ ID NO.135所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述牛痘病毒蛋白CPXV203的MHC结合结构域的氨基酸序列如SEQ ID NO.135所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述牛痘病毒蛋白CPXV203的KDEL受体结合结构域包含与SEQ ID NO.136所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述牛痘病毒蛋白CPXV203的KDEL受体结合结构域的氨基酸序列如SEQ ID NO.136所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述牛痘病毒蛋白CPXV203的全长序列包含与SEQ ID NO.137所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述牛痘病毒蛋白CPXV203的全长序列的氨基酸序列如SEQ ID NO.137所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述牛痘病毒蛋白CPXV203的MHC结合结构域包含与SEQ ID NO.138所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述牛痘病毒蛋白CPXV203的MHC结合结构域的氨基酸序列如SEQ ID NO.138所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述牛痘病毒蛋白CPXV203的KDEL受体结合结构域包含与SEQ ID NO.139所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述牛痘病毒蛋白CPXV203的KDEL受体结合结构域的氨基酸序列如SEQ ID NO.139所示。In some preferred embodiments, 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.
在一些实施方案中，所述多功能复合体还包含组件(4)复合型转接器(Chimeric adaptor)组件和/或靶向杀伤肿瘤细胞受体组件；In some embodiments, the multifunctional complex further includes component (4) Chimeric adapter component and/or targeted killing tumor cell receptor component;
所述(4)复合型转接器包含：(i)靶向肿瘤的胞外识别结构域；(ii)跨膜结构域；和(iii)胞内信号传导结构域；任选地，所述靶向肿瘤的胞外识别结构域、跨膜结构域和/或胞内信号结构域之间包含铰链或接头；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;
优选地，所述复合型转接器组件的靶向肿瘤的胞外识别结构域选自肿瘤抗原特异结合结构域、肿瘤微环境靶抗原结合结构域和/或靶向肿瘤微环境的趋化受体。Preferably, 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.
在一些优选的实施方案中，所述靶向肿瘤的胞外识别结构域选自能靶向识别肿瘤相关抗原的抗体或其功能片段、TCR或其组合；所述抗体的功能片段选自Fd、Fv、Fab、Fab’、F(ab’)2、Fv(scFv)、单链抗体(scFv)或纳米抗体(nanobody)、双链抗体、三链抗体和四链抗体。In some preferred embodiments, 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.
在一些优选的实施方案中，所述复合型转接器组件的跨膜结构域选自NK细胞激活受体跨膜结构域、DAP10跨膜结构域、DAP12跨膜结构域、CD8跨膜结构域、CD28跨膜结构域、CD4跨膜结构域、4-1BB跨膜结构域、OX40跨膜结构域、ICOS跨膜结构域、CTLA-4跨膜结构域、PD-1跨膜结构域、LAG-3跨膜结构域、2B4跨膜结构域和BTLA跨膜结构域以及其组合；优选地，所述NK细胞激活受体选自NKG2D、NKG2C、NKG2E、NKG2F、NKG2H、CD94、KIR2DL4、KIR2DS1、KIR2DS2、KIR2DS4、KIR3DS1，天然细胞毒性受体、TRAIL、DNAM-1、CD16a、2B4、NTB-A、CRACC以及NKp80；优选地，所述天然细胞毒性受体选自NKp46、NKp44和NKp30。In some preferred embodiments, 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, CD16a, 2B4, NTB-A, CRACC and NKp80; preferably, the natural cytotoxic receptor is selected from NKp46, NKp44 and NKp30.
在一些优选的实施方案中，所述复合型转接器组件的胞内信号传导结构域包括NK细胞激活受体的胞内信号结构域和/或共刺激信号传导结构域。In some preferred embodiments, 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.
在一些优选的实施方案中，所述NK细胞激活受体选自NKG2D、NKG2C、NKG2E、NKG2F、NKG2H、CD94、KIR2DL4、KIR2DS1、KIR2DS2、KIR2DS4、KIR3DS1、天然细胞毒性受体、TRAIL、DNAM-1、CD16a、2B4、NTB-A、CRACC以及NKp80。In some preferred embodiments, 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.
在一些优选的实施方案中，所述胞内信号结构域还包括共刺激信号传导结构域；优选地，所述共刺激信号传导结构域选自T细胞共刺激信号传导结构域；包括，但不限于衍生自MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白质、细胞因子受体、整联蛋白、淋巴细胞活化信号分子(SLAM蛋白)、活化NK细胞受体、BTLA、Toll配体受体、OX40、CD2、CD7、CD16、CD27、CD28、CD30、CD40、CD38、CD35、CD79A、CD79B、CDS、ICAM-1、LFA-1，(CD11a/CD18)、4-1BB(CD137)、B7-H3、CDS、ICAM-1、ICOS(CD278)、GITR、BAFFR、LIGHT、HVEM(LIGHTR)、KIRDS2、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、NKG2D、NKG2C、NCR、DAP10、DAP12、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD19a和与 CD83特异性结合的配体，CARD11、FcRa、FcRp、FcRy、Fyn、HVEM、ICOS、Lck、LAG3、LAT、LRP、NOTCH1、Wnt、OX40、ROR2、Ryk、SLAMF1、Slp76、pTa、TCRa、TCRp、TRIM、ZAP70、PTCH2等胞内信号结构域；更优选地，所述共刺激信号传导结构域选自NKG2D胞内信号结构域，DAP10胞内信号结构域，DAP12胞内信号结构域，NCR胞内信号结构域，CD28胞内信号结构域、4-1BB胞内信号结构域、OX40胞内信号结构域、ICOS胞内信号结构域；In some preferred embodiments, 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, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, NCR, DAP10, DAP12, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM , Ly9 (CD229), CD160 (BY55), PSGL1, CD100SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a and CD83 specific binding ligands, CARD11, FcRa, FcRp, FcRy, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, Wnt, OX40, ROR2, Ryk, SLAMF1, Slp76, pTa, TCRa, TCRp, TRIM, ZAP70, PTCH2 and other intracellular signaling domains; more preferably, the costimulatory signaling domain is selected from the group consisting of NKG2D intracellular signaling domain, DAP10 intracellular signaling domain, DAP12 intracellular signaling domain, and NCR intracellular signaling domain. Signaling domain, CD28 intracellular signaling domain, 4-1BB intracellular signaling domain, OX40 intracellular signaling domain, ICOS intracellular signaling domain;
所述靶向杀伤肿瘤细胞受体组件包含(i)靶向肿瘤抗原的胞外识别结构域；(ii)跨膜结构域；和(iii)胞内共刺激信号传导结构域；(iv)T细胞活化信号传导结构域(ITAM)；任选地，所述靶向肿瘤抗原的胞外识别结构域、跨膜结构域、胞内共刺激信号传导结构域和/或T细胞活化信号传导结构域(ITAM)之间包含铰链或接头；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;
所述靶向杀伤肿瘤细胞受体组件的跨膜结构域选自CD8跨膜结构域、T细胞受体的α和/或β链跨膜结构域、CD28跨膜结构域、CD3ε跨膜结构域、CD45跨膜结构域、CD4跨膜结构域、CD5跨膜结构域、CD8跨膜结构域、CD9跨膜结构域、CD16跨膜结构域、CD22跨膜结构域、CD33跨膜结构域、CD37跨膜结构域、CD64跨膜结构域、CD80跨膜结构域、CD86跨膜结构域、CD134跨膜结构域、CD137跨膜结构域、CD154跨膜结构域、GITR跨膜结构域以及其组合；The transmembrane domain targeting the tumor cell receptor component is selected from the group consisting of the CD8 transmembrane domain, the α and/or β chain transmembrane domain of the T cell receptor, the CD28 transmembrane domain, and the CD3ε 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;
所述T细胞活化信号传导结构域衍生自CD3ζ、共同FcRγ(FCER1G)、FcγRIIa、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD278(“ICOS”)、FcεRI CD66d、DAP10和DAP12等胞内信号结构域。The T cell activation signaling domains are derived from CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (“ICOS”), FcεRI CD66d, DAP10 and DAP12 and other intracellular signaling domains.
在一些优选的实施方案中，所述接头是柔性接头；优选地，所述柔性接头包含所示的氨基酸序列(Gly(x)Ser(y))n，其中n是1到10的整数，并且x和y独立地是0到10的整数，前提是x和y不都是0；更优选地，所述接头包含SEQ ID NO.140所示的氨基酸序列或SEQ ID NO.141所示的氨基酸序列。In some preferred embodiments, the linker is a flexible linker; preferably, the flexible linker comprises the amino acid sequence shown (Gly(x)Ser(y))n, 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.
在一些优选的实施方案中，所述接头是铰链；优选地，所述铰链是IgG1铰链或IgG4铰链。In some preferred embodiments, the linker is a hinge; preferably, the hinge is an IgG1 hinge or an IgG4 hinge.
在一些优选的实施方案中，所述IgG1铰链包含与SEQ ID NO.142所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述IgG1铰链的氨基酸序列如SEQ ID NO.142所示。In some preferred embodiments, the IgG1 hinge comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，所述IgG4铰链包含与SEQ ID NO.143所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述IgG4铰链的氨基酸序列如SEQ ID NO.143所示。In some preferred embodiments, the IgG4 hinge comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，所述NK激活受体组件、CNK信号转接组件和/或UT组件之间包含可切割肽；例如，T2A肽、GSG-T2A肽、E2A肽、GSG-E2A肽、F2A肽、GSG-F2A肽、P2A肽或GSG-P2A肽。In some preferred embodiments, 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.
在一些优选的实施方案中，所述T2A包含与SEQ ID NO.144所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述T2A的氨基酸序列如SEQ ID NO.144所示。In some preferred embodiments, 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.
在一些优选的实施方案中，GSG-T2A肽的氨基酸序列如SEQ ID NO.145所示。In some preferred embodiments, the amino acid sequence of the GSG-T2A peptide is as shown in SEQ ID NO. 145.
在一些优选的实施方案中，所述P2A包含与SEQ ID NO.146所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述P2A的氨基酸序列如SEQ ID NO.146所示。In some preferred embodiments, 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.
在一些优选的实施方案中，GSG-P2A肽的氨基酸序列如SEQ ID NO.147所示。In some preferred embodiments, the amino acid sequence of the GSG-P2A peptide is as shown in SEQ ID NO. 147.
在一些优选的实施方案中，所述E2A包含与SEQ ID NO.148所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述E2A的氨基酸序列如SEQ ID NO.148所示。In some preferred embodiments, 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.
在一些优选的实施方案中，GSG-E2A肽的氨基酸序列如SEQ ID NO.149所示。In some preferred embodiments, the amino acid sequence of the GSG-E2A peptide is as shown in SEQ ID NO. 149.
在一些优选的实施方案中，所述F2A包含与SEQ ID NO.150所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述F2A的氨基酸序列如SEQ ID NO.150所示。In some preferred embodiments, 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.
在一些优选的实施方案中，GSG-F2A肽的氨基酸序列如SEQ ID NO.151所示。In some preferred embodiments, the amino acid sequence of the GSG-F2A peptide is as shown in SEQ ID NO. 151.
在一些优选的实施方案中，所述多功能复合体包含与SEQ ID NO.152所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列包含如SEQ ID NO.152所示的TCR抗体单链抗体。In some preferred embodiments, 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.
在一些优选的实施方案中，所述多功能复合体包含与SEQ ID NO.153所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列如SEQ ID NO.153所示。In some preferred embodiments, 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.
在一方面，本公开提供了一种编码所述多功能复合体的核酸分子。In one aspect, the present disclosure provides a nucleic acid molecule encoding the multifunctional complex.
在一些优选的实施方案中，所述核酸分子是DNA或RNA。In some preferred embodiments, the nucleic acid molecule is DNA or RNA.
在一些优选的实施方案中，所述RNA是mRNA。In some preferred embodiments, the RNA is mRNA.
在一些优选的实施方案中，所述核酸分子包含与SEQ ID NO.154所示的核苷酸序列有80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或 99％以上同一性的氨基酸序列。In some preferred embodiments, 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.
在一方面，本公开提供了一种含有所述核酸的表达载体。In one aspect, the present disclosure provides an expression vector containing the nucleic acid.
在一些优选的实施方案中，所述载体选自质粒、粘粒、病毒载体、RNA载体或线性或圆形DNA或RNA分子。In some preferred embodiments, the vector is selected from plasmids, cosmids, viral vectors, RNA vectors or linear or circular DNA or RNA molecules.
在一些优选的实施方案中，所述病毒载体选自逆转录病毒、腺病毒、细小病毒(例如，腺伴随病毒)、冠状病毒、负链RNA病毒诸如正粘病毒(例如，流感病毒)、弹状病毒(例如，狂犬病和水疱性口炎病毒)、副粘病毒(例如，麻疼和仙台)、正链RNA病毒诸如小RNA病毒和甲病毒和双链DNA病毒，所述双链DNA病毒包括腺病毒、疱疹病毒(例如，单纯疱疹病毒1和2型、愛泼斯坦-巴尔病毒、巨细胞病毒)和痘病毒(例如，牛痘病毒、鸡痘病毒和金丝雀痘病毒)、诺沃克病毒、披膜病毒、黄病毒、呼肠孤病毒、乳多泡病毒、嗜肝DNA病毒、杆状病毒和肝炎病毒。In some preferred embodiments, 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, hepadnavirus, baculovirus and hepatitis virus.
在一些优选的实施方案中，所述逆转录病毒选自禽造白细胞组织增生-肉瘤、哺乳动物C-型、B-型病毒、D-型病毒、HTLV-BLV集合、慢病毒、泡沫病毒。In some preferred embodiments, 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.
在一些优选的实施方案中，所述慢病毒载体选自HIV-1、HIV-2、SIV、FIV、BIV、EIAV、CAEV或绵羊脱髓鞘性脑白质炎慢病毒。In some preferred embodiments, the lentiviral vector is selected from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or ovine demyelinating leukoencephalitis lentivirus.
在一些优选的实施方案中，所述NK激活受体组件、CNK信号转接组件和/或UT组件可在同一载体同一启动子，或不同启动子下调控表达，或多种载体中进行表达。In some preferred embodiments, 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.
在一些优选的实施方案中，所述载体为慢病毒载体，所述编码NK激活受体组件、CNK信号转接组件和/或UT组件的基因之间包含可切割肽编码基因；优选地，所述可切割肽为2A接头；所述2A接头选自T2A、P2A、E2A和F2A。In some preferred embodiments, 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.
在一些优选的实施方案中，所述载体还包含启动子；优选地，所述启动子为EF1α启动子或CMV启动子。In some preferred embodiments, the vector further comprises a promoter; preferably, the promoter is an EF1α promoter or a CMV promoter.
在一方面，本公开提供了一种免疫细胞，其包含所述的核酸或所述的表达载体。In one aspect, the present disclosure provides an immune cell comprising the nucleic acid or the expression vector.
在一些优选的实施方案中，免疫细胞选自T细胞，NKT细胞，NK细胞，B细胞，单核细胞，巨噬细胞等In some preferred embodiments, the immune cells are selected from T cells, NKT cells, NK cells, B cells, monocytes, macrophages, etc.
在一方面，本公开提供了一种制造免疫细胞的方法，其包括用选自下述的方法将所述的核酸或所述的表达载体引入细胞：电穿孔、声穿孔、基因枪(例如用Au-颗粒的基因枪)、脂质转染、聚合物转染、纳米颗粒或多聚复合体。In one aspect, 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.
在一方面，本公开提供了一种药物组合物，其包含的多功能复合体、所述的核酸、所述的表达载体、所述的免疫细胞和/或所述方法制造的免疫细胞，以及药学上可接受的载体。In one aspect, 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.
在一方面，本公开提供了一种多功能复合体、所述的核酸、所述的表达载体、所述的免疫细胞、所述方法制造的免疫细胞和/或所述的药物组合物在制备治疗疾病的药物中的用途。In one aspect, 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.
在一方面，本公开提供了一种治疗疾病的方法，其包括向受试者施用多功能复合体、所述的核酸、所述的表达载体、所述的免疫细胞和/或所述的药物组合物。In one aspect, 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.
在一些优选的实施方案中，所述疾病包括各类实体肿瘤和血液肿瘤，病毒感染性疾病，自身免疫性疾病。In some preferred embodiments, the diseases include various types of solid tumors and hematological tumors, viral infectious diseases, and autoimmune diseases.
在一些优选的实施方案中，所述实体肿瘤选自神经系统肿瘤，头颈部肿瘤，胸部肿瘤，消化系统肿瘤，泌尿生殖系统肿瘤，软组织和皮肤肿瘤，骨肿瘤等。In some preferred embodiments, 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.
在一些优选的实施方案中，神经系统肿瘤包括弥漫性胶质瘤，弥漫性星形细胞瘤和间变性星形细胞瘤，胶质母细胞瘤，少突胶质细胞瘤，少突星形细胞瘤，儿童弥漫性胶质瘤，其他星形细胞瘤，室管膜瘤，神经元和混合性神经元-神经胶质肿瘤，髓母细胞瘤，其他胚胎性肿瘤，神经鞘瘤，脑膜瘤，孤立性纤维性肿瘤和血管周细胞瘤等。In some preferred embodiments, 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.
在一些优选的实施方案中，头颈部肿瘤包括鼻腔及鼻窦恶性肿瘤，鼻咽癌，口腔癌，喉癌，涎腺肿瘤，颅内肿瘤，甲状腺癌，舌癌等。In some preferred embodiments, 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.
在一些优选的实施方案中，胸部肿瘤包括肺癌，食管癌，贲门癌，乳腺癌，纵膈肿瘤等。In some preferred embodiments, thoracic tumors include lung cancer, esophageal cancer, cardiac cancer, breast cancer, mediastinal tumors, and the like.
在一些优选的实施方案中，消化系统肿瘤包括胃癌，大肠癌及乙状结肠和直肠癌，肝癌，胰腺癌与壶腹周围癌，胆道癌，小肠恶性肿瘤等。In some preferred embodiments, digestive system tumors include gastric cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, small intestinal malignant tumors, and the like.
在一些优选的实施方案中，泌尿生殖系统肿瘤包括肾癌，前列腺癌，膀胱癌，睾丸恶性肿瘤，阴茎癌，子宫颈癌，子宫内膜癌，卵巢癌等。In some preferred embodiments, genitourinary tumors include kidney cancer, prostate cancer, bladder cancer, testicular malignancy, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, and the like.
在一些优选的实施方案中，软组织和皮肤肿瘤包括恶性纤维组织细胞瘤，横纹肌肉瘤，滑膜肉瘤，皮肤恶性黑色素瘤等。In some preferred embodiments, soft tissue and skin tumors include malignant fibrous histiocytoma, rhabdomyosarcoma, synovial sarcoma, cutaneous malignant melanoma, and the like.
在一些优选的实施方案中，骨肿瘤包括骨肉瘤，尤文氏肉瘤等。In some preferred embodiments, bone tumors include osteosarcoma, Ewing's sarcoma, and the like.
在一些优选的实施方案中，所述结肠癌是结肠腺瘤。In some preferred embodiments, the colon cancer is a colon adenoma.
在一些优选的实施方案中，所述乳腺癌是三阴乳腺癌细胞。In some preferred embodiments, the breast cancer is triple negative breast cancer cells.
在一些优选的实施方案中，所述肝癌是肝细胞癌。In some preferred embodiments, the liver cancer is hepatocellular carcinoma.
在一些优选的实施方案中，所述疾病是血液肿瘤，选自白血病、淋巴瘤(HL)、多发性骨髓瘤(MM)、骨髓增生异常综合症(MDS)等。In some preferred embodiments, the disease is a hematological neoplasm selected from the group consisting of leukemia, lymphoma (HL), multiple myeloma (MM), myelodysplastic syndrome (MDS), and the like.
在一些优选的实施方案中，所述白血病是B细胞急性淋巴性白血病，T细胞急性淋巴性白血病，急性髓系白血病等。 In some preferred embodiments, the leukemia is B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute myeloid leukemia, etc.
在一些优选的实施方案中，病毒感染性疾病包括：呼吸道病毒性疾病，胃肠道病毒性疾病，肝脏病毒性疾病，皮肤和黏膜病毒性疾病，眼病毒性疾病，中枢神经系统病毒性疾病，淋巴细胞性病毒性疾病，虫传病毒性疾病，慢病毒感染疾病等。In some preferred embodiments, 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.
在一些优选的实施方案中，呼吸道病毒性疾病包括鼻病毒、腺病毒、呼吸道合胞病毒、副流感病毒和冠状病毒等的感染；流行性感冒；流行性腮腺炎等。In some preferred embodiments, respiratory viral diseases include infections with rhinovirus, adenovirus, respiratory syncytial virus, parainfluenza virus, coronavirus, and the like; influenza; mumps, and the like.
在一些优选的实施方案中，胃肠道病毒性疾病包括脊髓灰质炎；库克萨基病毒感染；ECHO病毒感染；病毒性胃肠炎：包括轮状病毒性胃肠炎、诺瓦克病毒性胃肠炎、腺病毒性胃肠炎、星状病毒性胃肠炎、冠状病毒性胃肠炎和杯状病毒性胃肠炎等。In some preferred embodiments, 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.
在一些优选的实施方案中，肝脏病毒性疾病包括甲型病毒性肝炎、乙型病毒性肝炎、丙型病毒性肝炎、丁型病毒性肝炎、戊型病毒性肝炎、EB病毒性肝炎和巨细胞病毒性肝炎等。In some preferred embodiments, 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.
在一些优选的实施方案中，皮肤和黏膜病毒性疾病包括麻疹、风疹、幼儿急疹、水痘及带状疱疹、天花、单纯疱疹病毒感染、狂犬病和口蹄疫等。In some preferred embodiments, 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.
在一些优选的实施方案中，眼病毒性疾病包括流行性角膜结膜炎、滤泡性结膜炎和疱疹性角膜结膜炎等。In some preferred embodiments, ocular viral diseases include epidemic keratoconjunctivitis, follicular conjunctivitis, herpetic keratoconjunctivitis, and the like.
在一些优选的实施方案中，中枢神经系统病毒性疾病包括流行性乙型脑炎、西方马脑炎、东方马脑炎、圣路易脑炎、委内瑞拉马脑炎、墨累山谷脑炎、加利福尼亚脑炎、森林脑炎和淋巴细胞脉络丛脑膜炎等。In some preferred embodiments, 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.
在一些优选的实施方案中，淋巴细胞性病毒性疾病包括传染性单核细胞增多症、巨细胞病毒感染和获得性免疫缺陷综合征等。In some preferred embodiments, lymphocytic viral diseases include infectious mononucleosis, cytomegalovirus infection, acquired immunodeficiency syndrome, and the like.
在一些优选的实施方案中，虫传病毒性疾病包括病毒性出血热：包括流行性出血热、黄热病、克里米亚－刚果出血热、裂谷热、阿根廷出血热、玻利维亚出血热、拉萨热、鄂木斯克出血热、马尔堡病和埃波拉出血热等；登革热和登革出血热；西尼罗热；科罗拉多蜱传热；白蛉热等；In some preferred embodiments, the insect-borne viral diseases include viral hemorrhagic fevers: including epidemic hemorrhagic fever, yellow fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever, Omsk hemorrhagic fever, Marburg disease and Ebola hemorrhagic fever, etc.; Dengue fever and dengue hemorrhagic fever; West Nile fever; Colorado tick-borne fever; sand fly fever, etc.;
优选地，慢病毒感染疾病包括亚急性硬化性全脑炎、库鲁病、进行性多灶性白质脑病和亚急性海绵样脑病(皮质纹状体脊髓变性)等。Preferably, lentiviral infectious diseases include subacute sclerosing panencephalitis, kuru disease, progressive multifocal leukoencephalopathy, subacute spongiform encephalopathy (corticostriatal spinal cord degeneration), and the like.
在一些优选的实施方案中，自身免疫性疾病包括器官特异性自身免疫病和系统性自身免疫病；In some preferred embodiments, autoimmune diseases include organ-specific autoimmune diseases and systemic autoimmune diseases;
优选地，器官特异性自身免疫病包括慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症、急性特发性多神经炎等。Preferably, 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.
在一些优选的实施方案中，系统性自身免疫病包括系统性红斑狼疮、类风湿关节炎、系统性血管炎、硬皮病、天疱疮、皮肌炎、混合性结缔组织病、自身免疫性溶血性贫血、甲状腺自身免疫病、溃疡性结肠炎等。In some preferred embodiments, systemic autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune Hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, etc.
在一方面，本公开提供了一种刺激受试者中的免疫应答的方法，所述方法包括给予受试者有效量的多功能复合体、所述的核酸、所述的表达载体、所述的免疫细胞、所述方法制造的免疫细胞和/或所述的药物组合物。In one aspect, 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.
本公开还提供了细胞、细胞群以及包含所述细胞和群的组合物(包括药物和治疗组合物)，例如通过所提供的方法产生的细胞和群，以及方法，例如用于施用所述细胞和组合物至受试者如患者的治疗方法。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.
在一个实施方案中，所述促细胞凋亡蛋白为Bcl-2家族蛋白，包括但不限于Bax、Bak、Bok、Bad、Bid、Bik、Bim、HrkBnip3、Nix/Bnip3L、Noxa和Puma。In one embodiment, 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.
在一个实施方案中，本发明提供了一种嵌合蛋白构建体，其用于靶向特定蛋白质(即靶蛋白)以阻断其表达(例如，表面表达)并通过内质网相关降解(ER-associated degradation，ERAD)机制加速泛素化介导的降解。In one embodiment, 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.
内质网相关蛋白质降解(ERAD)是指靶向内质网中错误折叠的蛋白质进行泛素化并且随后通过蛋白酶体降解的细胞通路。ERAD的过程可分为三个步骤：(1)识别内质网中错误折叠或突变的蛋白质；(2)将识别的错误折叠或突变的蛋白质从内质网反向转运至细胞质中：末端错误折叠的蛋白质必须从内质网转运回细胞质中，而细胞质中包含泛素-蛋白酶体系统(ubiquitin–proteasome system，UPS)；(3)用蛋白酶体对识别的错误折叠或突变的蛋白质进行泛素依赖的降解(参见Annamaria et al.,ER-associated degradation:Protein quality control and beyond.J.Cell Biol.Vol.204No.6 869–879)。Endoplasmic reticulum-associated protein degradation (ERAD) refers to a cellular pathway that targets misfolded proteins in the endoplasmic reticulum for ubiquitination and subsequent degradation by the proteasome. The 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).
在这里，本申请提供了一种新的嵌合策略，将靶向蛋白结合结构域(比如特异性蛋白靶向抗体片段(ScFv))融合ERAD功能基序(比如病毒ER驻留蛋白TMD和ED返回信号域)，并且证明了该策略可以有效地抑制特定蛋白的表达(例如，特定蛋白在细胞表面的表达)。Here, 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).
本申请提供的TPD技术平台相比已有的蛋白降解/去除技术(例如，PROTAC、CRISPR等)具有以下优势：1.ER-TPD嵌合蛋白构建体通过载体(例如脂质体、纳米颗粒、慢病毒、腺病毒、溶瘤病毒等)递送至细胞内稳定表达发挥功能，相比PROTAC，不存在细胞通透性低和半衰期短导致降解效率低下的问题；2.ER-TPD技术基于内质网的ERAD原理，在内质网蛋白合成组装过程中直接干预蛋白合成并实现高效降解，在源头上可以实现对细胞膜表面蛋白和分泌型蛋白的降解，这是PROTAC无法靶向的蛋白，因此ER-TPD可以靶向大量常规技术无法靶向降级的蛋白；3.ER-TPD设计方式中，靶向结构域可以采用各类单链抗体结构或人工亲和配体结构，大大实现对目标蛋白的特异性识别和结合能力；而常规的PROTAC技术而常规的PROTAC技术采用小分子的化学结构很难保证靶向结构的特异性，因此ER-TPD技术具有靶向精确性和特异性的强大优势；4.ER-TPD技术可通过下游降解导向配体结构实现目标蛋白降解的多通路机制，包括基于泛素蛋白酶体的UPS机制和自噬溶酶体的ALP机制，下游降解导向配体结构设计可以通过基因工程方法，灵活机动的连接细胞内的降解系统，实现高效靶向降解，而常规 PROTAC技术只能通过靶向E3连接酶进行降解；5.TPD技术在蛋白层面降解目标蛋白，相比在基因层面阻断目标蛋白表达的技术(例如CRISPR技术、siRNA技术)而言不存在因脱靶效应或基因编辑过程而导致的染色体不稳定性等毒副作用；6.基于ER-TPD技术开发靶向降解药物的可行性高，周期短，同时基于ER-TPD技术的核酸和细胞药物生产成本低，更容易实现规模化和工业化。The TPD technology platform provided by this application 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. Compared with PROTAC, there is no problem of low cell permeability and short half-life leading to low degradation efficiency; 2. 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. It can degrade cell membrane surface proteins and secreted proteins at the source. This is a protein that PROTAC cannot target, so ER -TPD can target a large number of proteins that cannot be targeted and degraded by conventional technologies; 3. In the ER-TPD design method, 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 feasibility of developing targeted degradation drugs based on ER-TPD technology is high and the cycle is short. At the same time, the production cost of nucleic acid and cell drugs based on ER-TPD technology is low. , it is easier to achieve scale and industrialization.
在一方面，本公开提供了一种基于内质网的靶向蛋白降解技术(Endoplasmic Reticulum-based Targeted Protein Degradation，ER-TPD)的嵌合蛋白构建体(chimeric protein construct)，其包含内质网相关降解(ER-associated degradation，ERAD)机制蛋白结合结构域和靶向蛋白结合结构域。In one aspect, 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.
在某些实施方式中，所述嵌合蛋白构建体进一步包含蛋白降解通路成员(例如泛素-蛋白酶体系统通路成员、内体-溶酶体通路成员、自噬通路成员)结合结构域。所述蛋白降解通路成员结合结构域可以以任何合适的方式连接至所述嵌合蛋白构建体的组成元件，例如，与所述ERAD机制蛋白结合结构域连接。In certain embodiments, 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. 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.
在另一方面，本公开还提供了组合物文库，其中每种组合物包含病毒蛋白质或编码其的核酸分子，其包含能够劫持ERAD机制以在ER内阻滞并加速蛋白酶体介导的降解的功能基序和靶向特定蛋白质进行降解的亲和部分，为了治疗目的用于修饰细胞表型、改善细胞功能或诱导凋亡或抑制病毒复制。In another aspect, the present disclosure also provides a library of compositions, wherein each composition comprises a viral protein or a nucleic acid molecule encoding the same, which comprises a 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.
1.ERAD机制蛋白结合结构域1. ERAD mechanism protein binding domain
如在本文中所使用的术语“ERAD机制蛋白”指参与ERAD机制或通路的蛋白。术语“ERAD机制蛋白结合结构域”，也称为“ERAD降解结构域”，指能够结合和/或利用ERAD机制蛋白的部分或结构域，例如，病毒内质网(ER)驻留糖蛋白的跨膜结构域(或其功能变体)以及胞质结构域(或其功能变体)，该胞质结构域也称为“内质网驻留结构域”或“ER驻留结构域”。The term "ERAD mechanism protein" as used herein refers to a protein that participates in the ERAD mechanism or pathway. The term "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. a transmembrane domain (or a functional variant thereof) and a cytoplasmic domain (or a functional variant thereof), also known as the "endoplasmic reticulum resident domain" or "ER resident domain".
在一些实施方案中，所述ERAD机制蛋白结合结构域包含病毒内质网驻留蛋白的跨膜结构域或其功能变体和内质网驻留结构域或其功能变体。在一些实施方案中，病毒的ER驻留糖蛋白可以是任何病毒的ER驻留蛋白，它能够劫持ERAD机制并促进对靶蛋白的泛素化和蛋白酶体介导的降解。In some embodiments, the ERAD machinery protein binding domain comprises a transmembrane domain of a viral endoplasmic reticulum resident protein, or a functional variant thereof, and an endoplasmic reticulum resident domain, or a functional variant thereof. In some embodiments, the viral ER-resident glycoprotein can be any viral ER-resident protein that is capable of hijacking the ERAD machinery and promoting ubiquitination and proteasome-mediated degradation of the target protein.
i.衍生自腺病毒E3-19K的ERAD机制蛋白结合结构域i. ERAD machinery protein binding domain derived from adenovirus E3-19K
在一些实施方案中，所述病毒内质网驻留蛋白为腺病毒E3-19K。腺病毒E3-19K(也称为“E19”)包含三个功能模块：用于与MHC-I和MICA/B分子相互作用的腔域、跨膜结构域和内质网驻留结构域，其中其内质网驻留结构域包含细胞质尾中的二赖氨酸基序，该基序可将高尔基体返回到内质网，因此也可称为“ER返回信号基序”。研究表明，需要跨膜结构域和ER返回信号基序来确保有效的ER定位、组织相容性复合体I类(MHC-I)和MHC-I相关链A和B(MICA/B分子)的转运抑制以及蛋白酶体降解。因此，腺病毒E3-19K可以在分泌途径中驻留MHC I类分子(例如，MHC-I和MICA/B分子)并干扰抗原呈递。本公开提供的嵌合蛋白构建体利用前病毒E3-19K的跨膜结构域和内质网驻留结构域(即ERAD降解结构域)与靶向蛋白结合结构域融合，从而实现靶蛋白降解的目的。In some embodiments, the viral endoplasmic reticulum resident protein is adenovirus E3-19K. Adenovirus E3-19K (also known as "E19") contains three functional modules: a 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." Studies have shown that 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. Therefore, 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. 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.
在一些优选的实施方案中，所述腺病毒E3-19K的ERAD降解结构域包含与SEQ ID NO.96所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列。所述腺病毒E3-19K的ERAD降解结构域具有细胞质尾中的二赖氨酸基序，能够将高尔基体返回到内质网。在一些优选的实施方案中，所述腺病毒E3-19K的ERAD降解结构域的氨基酸序列如SEQ ID NO.96所示。In some preferred embodiments, 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. In some preferred embodiments, the amino acid sequence of the ERAD degradation domain of the adenovirus E3-19K is shown in SEQ ID NO. 96.
ii.衍生自其他病毒糖蛋白的ERAD机制蛋白结合结构域ii. ERAD machinery protein binding domains derived from other viral glycoproteins
在一些实施方案中，所述病毒内质网驻留蛋白不是腺病毒E3-19K。In some embodiments, the viral endoplasmic reticulum resident protein is not adenovirus E3-19K.
在一些实施方案中，所述病毒内质网驻留蛋白选自以下的至少一种：HCMV糖蛋白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和Cowpox Virus蛋白CPXV203。在一些实施方案中，所述病毒内质网驻留蛋白包含HCMV糖蛋白US2和US11。In some embodiments, the viral endoplasmic reticulum resident protein is selected from at least one of the following: HCMV glycoprotein US2, US11, US3, US10, US6, HSV ICP47, CPXV12, BHV UL49.5, EBV BNFL2a, HCMV UL16, UL141, UL142, HIV Nef, HIV Vpu, HHV-7U21, HHV-8KK3, HHV-8KK5, MHV-68MK3, HTLV-1p12 and Cowpox Virus protein CPXV203. In some embodiments, the viral endoplasmic reticulum resident protein comprises HCMV glycoproteins US2 and US11.
在一些实施方案中，所述病毒内质网驻留蛋白包含E3-19K以及选自以下的至少一种：HCMV糖蛋白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和Cowpox Virus蛋白CPXV203。In some embodiments, the viral endoplasmic reticulum resident protein comprises E3-19K and at least one selected from the group consisting of: HCMV glycoprotein US2, US11, US3, US10, US6, HSV ICP47, CPXV12, BHV UL49.5 , EBV BNFL2a, HCMV UL16, UL141, UL142, HIV Nef, HIV Vpu, HHV-7U21, HHV-8KK3, HHV-8KK5, MHV-68MK3, HTLV-1p12 and Cowpox Virus protein CPXV203.
a)HCMV US2和US11a)HCMV US2 and US11
HCMV US2和US11这两种蛋白都是ER驻留I型整合膜糖蛋白，它们共同选择该ERAD途径以促进MHC I类重链的降解，从而抑制MHC I类抗原呈递。两种蛋白中的任何一种蛋白质的表达都会导致新合成的MHC I类重链快速降解。US2和US11通过其腔结构域与MHC I类重链结合并募集宿主细胞蛋白，这些蛋白通过“拉动”重链的胞质尾部从内质网膜中提取多肽。MHC I类分子易位进入细胞质后，被泛素化，并被蛋白酶体降解。Both proteins, HCMV US2 and US11, are ER-resident type I integral membrane glycoproteins that jointly select this ERAD pathway to promote the degradation of MHC class I heavy chains, thereby inhibiting MHC class I antigen presentation. Expression of either protein results in rapid degradation of newly synthesized MHC class I heavy chains. US2 and US11 bind to MHC class I heavy chains through their luminal domains and recruit host cell proteins, 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.
除了I类分子外，US2还导致II类途径的两种蛋白质DR-α和DM-α以及HFE(一种参与铁调节的非经典主要组织相容性复合体(MHC)I类蛋白质)的降解。In addition to class I molecules, US2 leads to the degradation of two proteins of the class II pathway, DR-α and DM-α, and HFE, a nonclassical major histocompatibility complex (MHC) class I protein involved in iron regulation. .
US2的腔结构域负责结合MHC I类和II类分子，跨膜结构域(TM)和细胞质结构域(CT)与ERAD机制或途径的细胞成分相互作用，并有助于易位和促进MHC I类和II类这两种蛋白的酶解(Chevalier M S等人，2002，2003)。US2的胞质尾部足以与信号肽肽酶(SPP)相互作用，SPP是US2依赖性MHC I易位复合物(US2- dependent MHC I dislocation complex)的必要成分(Loureiro J et al.,2006)，对于US2依赖性MHC重链易位是必要的(参见Joana et al.,Signal peptide peptidase is required for dislocation from the endoplasmic reticulum.Nature volume 441,pages 894–897(2006))。此外，US2通过其TM结构域与内质网驻留RING型E3连接酶TRC8相互作用，这也有助于US2尾锚定MHC I和II分子的泛素化和蛋白酶体降解(Stagg H R et al.,2009)。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)). Furthermore, 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).
相比之下，US11诱导的MHC-I分子降解需要Derlin-1而不是SPP。US11的ER腔域与MHC-I重链的腔域相互作用，而US11的TM域与Derlin-1结合。因此，US11的主要功能可能是将MHC-I分子传递到Derlin-1(Lilley B N et al.,2004；Cho S et al.,2013)，然后诱导它们易位到胞质溶胶以进行蛋白酶体降解。此外，US11激活未折叠蛋白。通过Derlin-1，US11与作为ERAD RING E3连接酶的TMEM129相关联并招募Ube2J2在US11诱导降解之前泛素化MHC-I。In contrast, US11-induced degradation of MHC-I molecules requires Derlin-1 but not SPP. The ER luminal domain of US11 interacts with the luminal domain of 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. Furthermore, 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.
在一些优选的实施方案中，所述HCMV糖蛋白US2的ERAD降解结构域包含与SEQ ID NO.84所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述HCMV糖蛋白US2的ERAD降解结构域的氨基酸序列如SEQ ID NO.84所示。In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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. In some embodiments, the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US2 is shown in SEQ ID NO. 84.
在一些优选的实施方案中，所述US2的ERAD降解结构域包含与SEQ ID NO.112所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述US2的ERAD降解结构域的氨基酸序列如SEQ ID NO.112所示。In some preferred embodiments, 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. In some embodiments, the amino acid sequence of the ERAD degradation domain of US2 is shown in SEQ ID NO. 112.
在一些优选的实施方案中，所述HCMV糖蛋白US11的ERAD降解结构域包含与SEQ ID NO.90所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述HCMV糖蛋白US11的ERAD降解结构域的氨基酸序列如SEQ ID NO.90所示。In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US11 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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. In some embodiments, the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US11 is shown in SEQ ID NO. 90.
在一些优选的实施方案中，所述US11的ERAD降解结构域包含与SEQ ID NO.118所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述US11的ERAD降解结构域的氨基酸序列如SEQ ID NO.118所示。In some preferred embodiments, 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. In some embodiments, the amino acid sequence of the ERAD degradation domain of US11 is shown in SEQ ID NO. 118.
b)HCMV US3b)HCMV US3
HCMV US3糖蛋白不是促进MHC蛋白的降解，而是与载有肽的MHC I类异二聚体物理结合，导致I类复合物保留在ER中并抑制不变链与ER中II类DR-αβ二聚体的结合，导致II类复合物的错误定位和减少的肽负载。因此，在HCMV感染的早期阶段，US3能够干扰MHC I类分子的细胞内转运和成熟。US3是一种内质网驻留膜蛋白。结构域交换实验表明，US3的腔结构域足以使US3本身保持内质网，而腔内结构域和跨膜结构域对于内质网中I类MHC分子的保留都是必需的。Rather than promoting degradation of MHC proteins, 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-αβ 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. Domain exchange experiments showed that the luminal domain of US3 is sufficient for US3 itself to be retained in the endoplasmic reticulum, while both the luminal domain and the transmembrane domain are required for the retention of class I MHC molecules in the endoplasmic reticulum.
在一些优选的实施方案中，所述HCMV糖蛋白US3的ERAD降解结构域包含与SEQ ID NO.87所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述HCMV糖蛋白US3的ERAD降解结构域的氨基酸序列如SEQ ID NO.87所示。In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US3 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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. In some embodiments, the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US3 is shown in SEQ ID NO. 87.
在一些优选的实施方案中，所述US3的ERAD降解结构域包含与SEQ ID NO.115所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述US3的ERAD降解结构域的氨基酸序列如SEQ ID NO.115所示。In some preferred embodiments, 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. In some embodiments, the amino acid sequence of the ERAD degradation domain of US3 is shown in SEQ ID NO. 115.
c)HCMV US10c)HCMV US10
HCMV US10糖蛋白也与MHC I类抗原呈递的成分相互作用。US10结合游离的I类重链并延迟它们从ER的运输。但是，US10不影响US2或US11。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.
在一些优选的实施方案中，所述HCMV糖蛋白US10的ERAD降解结构域包含与SEQ ID NO.93所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述HCMV糖蛋白US10的ERAD降解结构域的氨基酸序列如SEQ ID NO.93所示。In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US10 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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. In some embodiments, the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US10 is shown in SEQ ID NO. 93.
d)HCMV US6、HSV ICP47、CPXV012、EBV BNFL2a、BHV UL49.5d)HCMV US6, HSV ICP47, CPXV012, EBV BNFL2a, BHV UL49.5
与US2、US3、US10和US11不同，HCMV L蛋白US6通过完全不同的策略影响抗原呈递。相反于与游离I类重链或完全组装的I类复合物相互作用，US6通过TAP复合物(TAP1/2)抑制胞质肽的易位。US6与TAP1的ER腔侧结合并导致构象变化，从而阻止ATP的结合。US6的ER-腔结构域中的残基89-108有助于US6与TAP的结合，并且是这种抑制的充分必要条件。这种对TAP活性的抑制不仅影响经典MHC I类等位基因的表达，还会影响胎儿细胞滋养层细胞中非经典等位基因HLA-C和HLA-G的表达。Unlike US2, US3, US10 and US11, HCMV L protein US6 affects antigen presentation through a completely different strategy. In contrast to interacting with free class I heavy chains or fully assembled class I complexes, US6 inhibits the translocation of cytosolic peptides through the TAP complex (TAP1/2). 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.
作为HCMV US6蛋白，HSV ICP47在感染周期的早期表达，对于体外复制是可有可无的，也可以应用相同的策略来阻止I类分子组装。ICP47阻断TAP介导的肽转运并与TAP1-TAP2复合物紧密结合。ICP47阻断TAP机制的一个线索是它表现出高物种选择性。HSV1和HSV2I CP47均抑制猿、猴、猪、狗和牛TAP，对小鼠、大鼠、豚鼠或兔TAP几乎没有影响。ICP47对人TAP的亲和力比对小鼠TAP的亲和力高约100倍。ICP47抑制肽与TAP的 结合，但不影响ATP结合。ICP47对TAP的亲和力比大多数肽高10-1000倍，可作为肽与TAP结合的竞争性抑制剂，并被认为直接与肽结合位点结合。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.
在一些优选的实施方案中，所述HHV-7US6的TAP结合结构域包含与SEQ ID NO.101所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述HHV-7US6的TAP结合结构域的氨基酸序列如SEQ ID NO.101所示。In some preferred embodiments, 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. In some embodiments, the amino acid sequence of the TAP binding domain of HHV-7US6 is shown in SEQ ID NO. 101.
在一些优选的实施方案中，所述HSV ICP47的TAP结合结构域包含与SEQ ID NO.103所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述HSV ICP47的TAP结合结构域的氨基酸序列如SEQ ID NO.103所示。In some preferred embodiments, the TAP binding domain of HSV ICP47 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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. In some embodiments, the amino acid sequence of the TAP binding domain of HSV ICP47 is shown in SEQ ID NO. 103.
在一些优选的实施方案中，所述CPXV012的TAP结合结构域包含与SEQ ID NO.105所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述CPXV012的TAP结合结构域的氨基酸序列如SEQ ID NO.105所示。In some preferred embodiments, 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. In some embodiments, the amino acid sequence of the TAP binding domain of CPXV012 is shown in SEQ ID NO. 105.
在一些优选的实施方案中，所述EBV BNFL2a的TAP结合结构域包含与SEQ ID NO.107所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述EBV BNFL2a的TAP结合结构域的氨基酸序列如SEQ ID NO.107所示。In some preferred embodiments, the TAP binding domain of EBV BNFL2a comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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. In some embodiments, the amino acid sequence of the TAP binding domain of EBV BNFL2a is shown in SEQ ID NO. 107.
在一些优选的实施方案中，所述BHV UL49.5的TAP结合结构域包含与SEQ ID NO.109所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述BHV UL49.5的TAP结合结构域的氨基酸序列如SEQ ID NO.109所示。In some preferred embodiments, 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. In some embodiments, the amino acid sequence of the TAP binding domain of BHV UL49.5 is shown in SEQ ID NO. 109.
e)其他e)Others
在一些优选的实施方案中，所述HHV-7US21的ERAD降解结构域包含与SEQ ID NO.99所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述HHV-7US21的ERAD降解结构域的氨基酸序列如SEQ ID NO.99所示。In some preferred embodiments, 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. In some embodiments, the amino acid sequence of the ERAD degradation domain of HHV-7US21 is shown in SEQ ID NO. 99.
在一些优选的实施方案中，所述HCMV UL16的ERAD降解结构域包含与SEQ ID NO.121所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述HCMV UL16的ERAD降解结构域的氨基酸序列如SEQ ID NO.121所示。In some preferred embodiments, the ERAD degradation domain of HCMV UL16 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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. In some embodiments, the amino acid sequence of the ERAD degradation domain of HCMV UL16 is shown in SEQ ID NO. 121.
在一些优选的实施方案中，所述HCMV UL141的ERAD降解结构域包含与SEQ ID NO.124所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述HCMV UL141的ERAD降解结构域的氨基酸序列如SEQ ID NO.124所示。In some preferred embodiments, the ERAD degradation domain of HCMV UL141 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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. In some embodiments, the amino acid sequence of the ERAD degradation domain of HCMV UL141 is shown in SEQ ID NO. 124.
在一些优选的实施方案中，所述HCMV UL142的高尔基驻留结构域包含与SEQ ID NO.127所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述HCMV UL142的高尔基驻留结构域的氨基酸序列如SEQ ID NO.127所示。In some preferred embodiments, 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. In some embodiments, the amino acid sequence of the Golgi-resident domain of HCMV UL142 is set forth in SEQ ID NO. 127.
在一些优选的实施方案中，所述HIV Nef包含与SEQ ID NO.128所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述HIV Nef的氨基酸序列如SEQ ID NO.128所示。In some preferred embodiments, the HIV Nef comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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. In some embodiments, the amino acid sequence of HIV Nef is set forth in SEQ ID NO. 128.
在一些优选的实施方案中，所述HIV Vpu包含与SEQ ID NO.129所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述HIV Vpu的氨基酸序列如SEQ ID NO.129所示。In some preferred embodiments, the HIV Vpu comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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. In some embodiments, the HIV Vpu has an amino acid sequence as shown in SEQ ID NO. 129.
在一些优选的实施方案中，所述HHV-8KK3包含与SEQ ID NO.130所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述HHV-8KK3的氨基酸序列如SEQ ID NO.130所示。In some preferred embodiments, 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. In some embodiments, the amino acid sequence of HHV-8KK3 is shown in SEQ ID NO. 130.
在一些优选的实施方案中，所述HHV-8KK5包含与SEQ ID NO.131所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述HHV-8KK5的氨基酸序列如SEQ ID NO.131所示。 In some preferred embodiments, 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. In some embodiments, the amino acid sequence of HHV-8KK5 is set forth in SEQ ID NO. 131.
在一些优选的实施方案中，所述MHV-68MK3包含与SEQ ID NO.132所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述MHV-68MK3的氨基酸序列如SEQ ID NO.132所示。In some preferred embodiments, 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. In some embodiments, the amino acid sequence of MHV-68MK3 is shown in SEQ ID NO. 132.
在一些优选的实施方案中，所述HTLV-1p12包含与SEQ ID NO.133所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述HTLV-1p12的氨基酸序列如SEQ ID NO.133所示。In some preferred embodiments, 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. In some embodiments, the amino acid sequence of HTLV-1p12 is shown in SEQ ID NO. 133.
在一些优选的实施方案中，所述牛痘病毒蛋白CPXV203的KDEL受体结合结构域包含与SEQ ID NO.136所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述牛痘病毒蛋白CPXV203的KDEL受体结合结构域的氨基酸序列如SEQ ID NO.136所示。In some preferred embodiments, 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. In some embodiments, the amino acid sequence of the KDEL receptor binding domain of the vaccinia virus protein CPXV203 is shown in SEQ ID NO. 136.
在一些优选的实施方案中，所述牛痘病毒蛋白CPXV203的KDEL受体结合结构域包含与SEQ ID NO.139所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，并具有内质网驻留的功能。在一些实施方案中，所述牛痘病毒蛋白CPXV203的KDEL受体结合结构域的氨基酸序列如SEQ ID NO.139所示。In some preferred embodiments, 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. In some embodiments, the amino acid sequence of the KDEL receptor binding domain of the vaccinia virus protein CPXV203 is shown in SEQ ID NO. 139.
2.靶向结构域2. Targeting domain
本公开提供的嵌合蛋白构建体的靶向结构域(target binding domain)可以是识别并结合靶蛋白的任何结构，例如抗体、抗体片段、从天然蛋白衍生的功能基序(例如，天然配体)、人工合成的对靶蛋白有亲和力的多肽或蛋白，或者上述分子的突变体、融合体、截短体等各种变体形式。例如，靶向结构域可以为靶蛋白的天然配体、特异性识别靶蛋白的抗体或其抗原结合片段，也可以是能够被抗体特异性识别的抗原。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. For example, 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.
在一些实施方案中，所述靶向结构域包含特异性靶向靶蛋白的抗体或其功能片段(例如，双功能抗体、Fab、Fab'、F(ab')2、Fd、Fv片段、二硫键稳定的Fv片段(dsFv)、(dsFv)2、双特异性dsFv(dsFv-dsFv')、二硫键稳定的双功能抗体(ds双功能抗体)、单链抗体分子(scFv)、scFv二聚体(二价双功能抗体)、Fv(scFv)、多特异性抗体(例如，双特异性抗体)、骆驼化单结构域抗体(例如，VHH)、纳米抗体、结构域抗体、二价结构域抗体、双链抗体、三链抗体和四链抗体。所述靶向蛋白结构域还可以包含任何能够结合靶蛋白的结合配体，包括但不限于，配体、受体中结合配体的部分、病毒蛋白中结合宿主蛋白的部分等。In some embodiments, 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 host protein, etc.
靶蛋白可以是其水平或活性期望被调节的任何蛋白，例如，通过ERAD机制改变活性以阻滞ER中的蛋白质并加速蛋白酶体介导的降解，其降解可以改变细胞的表型、提高细胞功能、引起细胞凋亡和/或抑制病毒复制等，从而起到治疗性目的(例如，提高疗效、降低副作用等)。在一些实施方案中，靶蛋白可以是参与细胞周期、凋亡、信号转导、细胞分化、细胞去分化、细胞生长、细胞因子或其生物调节剂的产生、细胞因子或生物调节剂的产生、调节或功能蛋白的产生、促炎信号传导或葡萄糖调节途径的蛋白。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.). In some embodiments, 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.
在另一个实施方案中，靶蛋白是疾病相关蛋白(也称为“致病蛋白”)，本文中所述的“疾病相关蛋白”或“致病蛋白”指任何其功能或活性的改变将导致疾病的发生，或者其功能对于疾病的发展而言比较重要的蛋白，示例性致病蛋白包括但不限于致癌蛋白、病毒蛋白、致自身免疫性疾病蛋白(例如，浆细胞产生的抗体)。In another embodiment, the target protein is a disease-associated protein (also referred to as a "pathogenic protein"), and as used herein, "disease-associated protein" or "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. Examples of pathogenic proteins include, but are not limited to, oncogenic proteins, viral proteins, and autoimmune disease-causing proteins (for example, antibodies produced by plasma cells).
在一些实施方案中，所述靶蛋白选自：免疫功能相关的靶蛋白、神经系统疾病相关的靶蛋白、感染类疾病相关的靶蛋白(例如病毒感染类相关的靶蛋白)、自身抗原相关的靶蛋白或肿瘤相关的靶蛋白(或致癌蛋白)、代谢疾病相关的靶蛋白。In some embodiments, 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.
在一些实施方案中，所述致病蛋白为致癌蛋白。所述致癌蛋白可以由致癌基因编码，其包括但不限于BCL2、c-MYC、Ras、HER2、BCR/ABL、ABL1/BCR、TGFB1、TLX1、P53、WNT1、WNT2、WT1、αv-β3、PKCa、ABL、BCL1、CD24、CDK4、EGFR/ERBB-1、HSTF1、INT1/WNT1、INT2、MDM2、MET、MYB、MYC、MYCN、MYCL1、RAFI、NRAS、REL、AKT2、APC、BCL2-ALPHA、BCL2-BETA、BCL3、BCR、BRCA1、BRCA2、CBL、CCND1、CDKN1A、CDKN1C、CDKN2A、CDKN2B、CRK、CRK-II、CSF1R/FMS、DBL、DDOST、PMS-2、PRAD-1、RAF、RHOM-1、RHOM-2、SIS、TAL2、TANI、TIAM1、TSC2、TRK、TSC1、STK11、PTCH、MEN1、MEN2、P57/KIP2、PTEN、HPC1、ATM、XPA/XPG、BCL6、DEK、AKAP13、CDH1、BLM、EWSR1/FLI1、FES、FGF3、FER、FGR、FLI1/ERGB2、FOS、FPS/FES、FRA1、FRA2、FYN、HCK、HEK、HER3/ERBB-2、ERBB-3、HER4/ERBB-4、HST2、INK4A、INK4B、JUN、JUNB、JUND、KIP2、KIT、KRAS2A、KRAS2B、LCK、LYN、MAS、MAX、MCC、MLH1、MOS、MSH2、MYBA、MYBB、NF1、NF2、P53、PDGFB、PIM1、PTC、RBI、RET、ROS1、SKI、SRC1、TALI、TGFBR2、THRA1、THRB、TIAM1、TRK、VAV、VHL、WAF1、WNT2、WT1、YES1、ALK/NPM1、AMI1、AXL、FMS、GIP、GLI、GSP、HOX11、HST、IL3、INT2、KS3、K-SAM、LBC、DCC、DPC4/SMAD4、E-CAD、E2F1/RBAP、ELK1、ELK3、EPH、EPHA1、E2F1、EPHA3、ERG、ETS1、ETS2、LMO-1、LMO-2、L-MYC、LYL1、LYT-10、MDM-2、MLH1、MLL、MLM、N-MYC、OST、PAX-5、PMS-1、FGF4、FGF6、FANCA、FLI1/ERGB2、FOSL1、FOSL2、GLI、HRAS1、HRX/MLLT1、HRX/MLLT2、KRAS2、MADH4、MASI、MCF2、MLLT1/MLL、MLLT2/HRX、MTG8/RUNX1、MYCLK1、MYH11/CBFB、NFKB2、NOTCH1、NPM1/ALK、 NRG/REL、NTRK1、PBX1/TCF3、PML/RARA、PRCA1、RUNX1、RUNX1/CBFA2T1、SET、SHP2、TCF3/PBX1、TNFa、Clusterin、Survivin、ΤΟΕβ、c-fos,c-SRC和INT-1.在某些实施方式中，所述致癌蛋白可以是Bcl-2家族成员(如：Bcl-2,Bcl-xL and Bcl-w)、VEGF/VEGFR、PDGFRβ、EGFR、EGFR突变体、IGF-1R、HDACs、HER2、MYC、KRAS、AFP,CEA,CA199,雌激素受体(estrogen receptor ER-α)、雄激素受体(Androgen receptor,AR)、酪氨酸激酶(c-ABL、BCR-ABL、BTK、FAK、PTK6、Wee1、TRK跨膜受体)、丝/苏氨酸激酶受体(IRAK4、LRRK2、B-Raf、RIPK2、CDK4/6、CDK7、CDK8、CDK8/19、CDK9、TBK1)、蛋白激酶Ⅱ(CK2)、表观遗传相关蛋白(BRD2、BRD3、BRD4、BRDT、TRIM24、BRD9、PBRM1、SMARCA2、SMARCA4、EP300、EZH2、WDR5)、肾上腺髓质素(ADM)、DPP3。In some embodiments, 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, αv-β3, PKCa , ABL, BCL1, CD24, CDK4, EGFR/ERBB-1, HSTF1, INT1/WNT1, INT2, MDM2, MET, MYB, MYC, MYCN, MYCL1, RAFI, NRAS, REL, AKT2, APC, BCL2-ALPHA, BCL2 -BETA, BCL3, BCR, BRCA1, BRCA2, CBL, CCND1, CDKN1A, CDKN1C, CDKN2A, CDKN2B, CRK, CRK-II, CSF1R/FMS, DBL, DDOST, PMS-2, PRAD-1, RAF, RHOM-1 , RHOM-2, SIS, TAL2, TANI, TIAM1, TSC2, TRK, TSC1, STK11, PTCH, MEN1, MEN2, P57/KIP2, PTEN, HPC1, ATM, XPA/XPG, BCL6, DEK, AKAP13, CDH1, BLM , EWSR1/FLI1, FES, FGF3, FER, FGR, FLI1/ERGB2, FOS, FPS/FES, FRA1, FRA2, FYN, HCK, HEK, HER3/ERBB-2, ERBB-3, HER4/ERBB-4, HST2 , INK4A, INK4B, JUN, JUNB, JUND, KIP2, KIT, KRAS2A, KRAS2B, LCK, LYN, MAS, MAX, MCC, MLH1, MOS, MSH2, MYBA, MYBB, NF1, NF2, P53, PDGFB, PIM1, PTC , RBI, RET, ROS1, SKI, SRC1, TALI, TGFBR2, THRA1, THRB, TIAM1, TRK, VAV, VHL, WAF1, WNT2, WT1, YES1, ALK/NPM1, AMI1, AXL, FMS, GIP, GLI, GSP , HOX11, HST, IL3, INT2, KS3, K-SAM, LBC, DCC, DPC4/SMAD4, E-CAD, E2F1/RBAP, ELK1, ELK3, EPH, EPHA1, E2F1, EPHA3, ERG, ETS1, ETS2, LMO -1, LMO-2, L-MYC, LYL1, LYT-10, MDM-2, MLH1, MLL, MLM, N-MYC, OST, PAX-5, PMS-1, FGF4, FGF6, FANCA, FLI1/ERGB2 , FOSL1, FOSL2, GLI, HRAS1, HRX/MLLT1, HRX/MLLT2, KRAS2, MADH4, MASI, MCF2, MLLT1/MLL, MLLT2/HRX, MTG8/RUNX1, MYCLK1, MYH11/CBFB, NFKB2, NOTCH1, NPM1/ALK , NRG/REL, NTRK1, PBX1/TCF3, PML/RARA, PRCA1, RUNX1, RUNX1/CBFA2T1, SET, SHP2, TCF3/PBX1, TNFa, Clusterin, Survivin, ΤΟΕβ, c-fos, c-SRC and INT-1. In certain embodiments, the oncogenic protein can be a Bcl-2 family member (such as: Bcl-2, Bcl-xL and Bcl-w), VEGF/VEGFR, PDGFRβ, EGFR, EGFR mutants, IGF-1R, HDACs, HER2, MYC, KRAS, AFP, CEA, CA199, estrogen receptor (estrogen receptor ER-α), 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, PBRM1, SMARCA2, SMARCA4, EP300, EZH2, WDR5), adrenomedullin (ADM), DPP3.
在一些实施方案中，所述靶蛋白包括免疫功能相关蛋白。如本文中所使用的术语“免疫功能相关蛋白”指参与机体免疫过程的功能蛋白，例如抗原呈递分子(例如MHC I类分子、MHC II类分子、HLA等)、抗原识别分子(例如TCR、CD123、NKG2D等)、免疫检查点分子(例如PD-1、PD-L1、CTLA4、TIM3,TIGIT,LAG3,A2AR,BTLA,IDO1,IDO2,TDO,KIR,NOX2,VISTA,SIGLEC7,PVR等)、免疫刺激/共刺激分子(例如CD3、CD80/86、CD28等)，以及其他参与先天性或适应性免疫的分子。在一些实施方案中，免疫功能相关的靶蛋白包括，例如CD123、CD7、CD5、MHC I类分子、MHC II类分子、非经典MHC分子(如HLA-G,HLA-E)，MICA/B、ULBP1-6、IL6、IL6受体、IL1受体、RANKL、TGF-β1、PD1、PD-L1、CTLA4、Tim3、LAG3、Siglec-15、TIGIT、CD47、IL4RA、CD94/NKG2A、CXCR1/2、CXCL8、CCR2/CCR5、CCR4、CXCR4、c-Rel、CCL2、CCL5、CCL20、CCL22、CSF-1、CCL2、CCL5吲哚胺-2,3-二加氧酶(IDO)、或精氨酸酶1(ARG1)。In some embodiments, the target protein includes an immune function-related protein. As used herein, the term "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. CD3, CD80/86, CD28, etc.), and other molecules involved in innate or adaptive immunity. In some embodiments, immune function-related target proteins include, for example, CD123, CD7, CD5, MHC class I molecules, MHC class II molecules, non-classical MHC molecules (such as HLA-G, HLA-E), MICA/B, ULBP1-6, IL6, IL6 receptor, IL1 receptor, RANKL, TGF-β1, PD1, PD-L1, CTLA4, Tim3, LAG3, Siglec-15, TIGIT, CD47, IL4RA, CD94/NKG2A, CXCR1/2, CXCL8, CCR2/CCR5, CCR4, CXCR4, c-Rel, CCL2, CCL5, CCL20, CCL22, CSF-1, CCL2, CCL5 indoleamine-2,3-dioxygenase (IDO), or arginase 1(ARG1).
在一些实施方案中，所述靶蛋白包括神经系统疾病相关的靶蛋白。如本文中所使用的术语“神经系统疾病相关”指所述蛋白参与神经系统疾病，特别是在神经退行性疾病中的蛋白。在一些实施方案中，所述神经系统疾病相关的靶蛋白包括，例如，Tau、amyloid-β(Aβ)、α突触核蛋白、亨廷顿蛋白突变体(mutant huntingtin,mHTT)、α-synuclein、TAR RNA结合蛋白(TARDBP)和FUS RNA结合蛋白(FUS)。In some embodiments, 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. In some embodiments, the target protein associated with neurological disease includes, for example, Tau, amyloid-β (Aβ), alpha synuclein, mutant huntingtin (mHTT), alpha-synuclein, TAR RNA binding protein (TARDBP) and FUS RNA binding protein (FUS).
在一些实施方案中，所述靶蛋白包括病毒感染相关的靶蛋白。所述病毒感染相关的靶蛋白包括，例如，HBV编码X蛋白(HBx)、HBV DNA聚合酶、HBV衣壳糖蛋白、HIV-1逆转录酶、HIV gp120、HCV NS3-4A蛋白酶、HCV RNA聚合酶、HCV包膜(envelope)蛋白、EBV DNA聚合酶、EBV EBNA1、冠状病毒RNA合成酶、冠状病毒刺突蛋白、冠状病毒包膜蛋白、冠状病毒膜蛋白、冠状病毒核壳蛋白、RNA依赖性RNA聚合酶(RNA-dependent RNA polymerase,RdRp)，如新冠病毒RNA依赖性RNA聚合酶、疱疹病毒(Herpesviruses)DNA和RNA聚合酶、疱疹病毒衣壳糖蛋白、CMV DNA聚合酶、CMV衣壳糖蛋白、RSV表膜蛋白、RSV衣壳蛋白、RSV RNA聚合酶、流感病毒RNA聚合酶、流感病毒包膜(envelope)蛋白、HPV DNA聚合酶、HPV衣壳蛋白。在另一个实施方案中，靶蛋白是病毒蛋白，例如HBV表面抗原、HBeAg、HBV聚合酶蛋白、HIV Gag蛋白、HIV Env蛋白等，其功能被认为对病毒复制扩增和功能以及病毒性疾病的进展重要。In some embodiments, 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 RNA polymerase, influenza virus envelope protein, HPV DNA polymerase, HPV capsid protein. In another embodiment, the target protein is a viral protein, such as HBV surface antigen, HBeAg, HBV polymerase protein, HIV Gag protein, HIV Env protein, etc., the function of which is believed to be important for viral replication amplification and function as well as viral diseases. Progress matters.
在一些实施方案中，所述靶蛋白包括自身抗原相关的靶蛋白。本领域已知多种与自身免疫疾病相关的自身抗原。例如，与I型糖尿病相关的自身抗原例如，胰岛细胞抗原(ICA)、胰岛素(IAA)、谷氨酸脱羧酶65(GAD65)，胰岛瘤抗原-2(IA-2)；与类风湿性关节炎(RA)相关的自身抗原(autoantigen)例如，瓜氨酸化蛋白/肽抗体，异核糖核蛋白(heterogeneous nuclear ribonucleoprotein A2/B1),aldolase,alpha-enolase,calreticulin,热激活蛋白(HSP60)、BiP、PGK1、压力诱导磷酸蛋白(stress-induced phosphoprotein 1)、FUSE-BP1/2；与系统性红斑狼疮(SLE)相关的自身抗原例如，脱氧核糖核蛋白，SmD1和SmD3，Clq，疮抗凝物(LA)、心磷脂(CL)、β2糖蛋白I(β2GP I)、凝血酶原(PT)和磷脂酰丝氨酸(PS)；与系统性硬化症(Systemic Sclerosis，SSc)/硬皮病(scleroderma SD)相关自身抗原例如，Scl-70、SSA、Ro52；与自身免疫性肝病相关的抗原例如，线粒体抗原、Spl00、PML、gp210、p62；与重症肌无力相关的自身抗原例如，乙酰胆碱受体；与中枢神经系统自身免疫疾病(边缘性脑炎、脑脊髓炎、小脑性共济失调)相关的自身抗原例如，电压门控性钾离子通道(VGKC)复合物、电压门控性钙离子通道受体、a-氨基-3-羟基-5-甲基异噁唑-4-丙酸(AMPA)受体、γ氨基丁酸-B(GABAB)受体、甘氨酸受体；与多发性硬化相关的自身抗原例如，髓磷脂碱性蛋白(MBP)、髓磷脂少突胶质细胞糖蛋白(MOG)；与多发性肌炎(PM)和皮肌炎(DM)相关的自身抗原例如，Jo-1、Mi-2、PM-Scl、Ro-52；与麸质敏感性肠病相关的自身抗原例如，肌内膜抗体(EMA)、组织谷氨酰胺转移酶(tTG)；与抗NMDAR抗体脑炎相关的自身抗原例如，N-甲基-D-天冬氨酸受体；与视神经脊髓炎(NMO)相关的自身抗原例如，水通道蛋白4(AQP4)；与生殖相关相关的自身抗原例如，卵巢抗原，精子抗原。In some embodiments, the target protein includes an autoantigen-related target protein. A variety of autoantigens associated with autoimmune diseases are known in the art. For example, autoantigens associated with type I diabetes 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), β2 glycoprotein I (β2GP I), prothrombin (PT) and phosphatidylserine (PS); and systemic sclerosis (SSc)/scleroderma (scleroderma) SD) related autoantigens such as Scl-70, SSA, Ro52; antigens related to autoimmune liver disease such as mitochondrial antigens, Spl00, PML, gp210, p62; autoantigens related to myasthenia gravis such as acetylcholine receptor; Autoantigens associated with central nervous system autoimmune diseases (limbic encephalitis, encephalomyelitis, cerebellar ataxia) include voltage-gated potassium channel (VGKC) complex, voltage-gated calcium channel receptor body, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor, gamma-aminobutyric acid-B (GABAB) receptor, glycine receptor; associated with multiple sclerosis Autoantigens such as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG); autoantigens associated with polymyositis (PM) and dermatomyositis (DM) such as Jo-1 , Mi-2, PM-Scl, Ro-52; autoantigens associated with gluten-sensitive enteropathy such as endomysial antibodies (EMA), tissue transglutaminase (tTG); and anti-NMDAR antibodies in encephalitis Relevant autoantigens such as N-methyl-D-aspartate receptor; autoantigens associated with neuromyelitis optica (NMO) such as aquaporin 4 (AQP4); autoantigens associated with reproduction such as, Ovarian antigen, sperm antigen.
在一些实施方案中，所述致病蛋白为代谢疾病相关的靶蛋白。所述代谢疾病相关的靶蛋白例如，关于动脉粥样硬化(Atherosclerosis，AS)的靶蛋白，包括但不限于，CD36、低密度脂蛋白受体(LDLR)、ChemR23(CMKLR1)、线粒体脱氢酶(mitochondrial dehydrogenase)ALDH4A1(的靶蛋)；关于2型糖尿病的靶蛋白，包括但不限于，RalGAPα1、二肽基肽酶Ⅳ(DPP4)；关于非酒精性脂肪肝的靶蛋白，包括但不限于，TMEM16A、VAMP3；关于肿瘤糖代谢的靶蛋白，包括但不限于，己糖激酶(hexokinase，HK)、葡萄糖转运体1(glucosetransporter 1，GLUT1)、葡萄糖转运体4(glucose transporter 4，GLUT4)、磷酸甘油酸脱氢酶、乳酸脱氢酶；关于肿瘤脂代谢的靶蛋白，包括但不限于，ATP柠檬酸裂解酶(ATP citrate lyase，ACLY)、脂肪酸合成酶(fatty acid synthase，FASN)、胆固醇酯化酶(acetyl-CoA acetyltransferase 1，ACAT1)。In some embodiments, 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, RalGAPα1, 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 lyase (ACLY), fatty acid synthase (FASN), cholesterol Esterase (acetyl-CoA acetyltransferase 1, ACAT1).
在一些实施方案中，靶向结构域特异性靶向除了以下靶蛋白的其他靶蛋白：TCR、HLA-I、MICA和MICB， 且所述病毒内质网驻留蛋白是本公开所述的任何病毒内质网驻留蛋白。In some embodiments, the targeting domain specifically targets target proteins other than TCR, HLA-I, MICA, and MICB, And the viral endoplasmic reticulum resident protein is any viral endoplasmic reticulum resident protein described in the present disclosure.
在一些实施方案中，靶向结构域特异性靶向至少2个以下靶蛋白：TCR、HLA-I、MICA、MICB，所述病毒内质网驻留蛋白是本公开所述的任何病毒内质网驻留蛋白。In some embodiments, 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.
在一些实施方案中，靶向结构域特异性靶向TCR、HLA-I、MICA或MICB，且所述病毒内质网驻留蛋白不是腺病毒E3-K19。In some embodiments, the targeting domain specifically targets TCR, HLA-I, MICA, or MICB, and the viral endoplasmic reticulum resident protein is not adenovirus E3-K19.
在一些实施方式中，所述嵌合蛋白构建体的靶向结构域通过铰链或连接子与ERAD机制蛋白结合结构域相连。在一些实施方式中，所述铰链包含如SEQ ID NO:143(IgG4铰链)或SEQ ID NO:141((Gly4Ser)2)所示的氨基酸序列。In some embodiments, the targeting domain of the chimeric protein construct is connected to the ERAD machinery protein binding domain via a hinge or linker. In some embodiments, the hinge comprises the amino acid sequence set forth in SEQ ID NO: 143 (IgG4 hinge) or SEQ ID NO: 141 ((Gly4Ser)2).
3.蛋白降解通路成员结合结构域3. Binding domain of protein degradation pathway members
本公开提供的嵌合蛋白构建体还可以包含蛋白降解通路成员结合结构域。如本文所述的“蛋白降解通路成员结合结构域”是指任何能够与蛋白降解通路中的成员直接或间接结合的部分。The chimeric protein constructs provided by the present disclosure may also include protein degradation pathway member binding domains. As used herein, "protein degradation pathway member binding domain" refers to any part that can directly or indirectly bind to a member of a protein degradation pathway.
蛋白降解通路可以是任何能够介导蛋白在细胞内降解的通路。已知的蛋白降解通路包括，但不限于，基于泛素化-蛋白酶体通路，内体-溶酶体通路，和自噬降解通路等。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.
泛素化-蛋白酶体通路成员包括例如，E1泛素激活酶、E2泛素结合酶、E3泛素连接酶、和蛋白酶体。Members of the ubiquitination-proteasome pathway include, for example, E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, E3 ubiquitin ligases, and the proteasome.
E1泛素激活酶的例子包括，例如UBA1、UBA2、UBA3、UBA5、UBA6、UBA7、ATG7、NAE1、和SAE1。Examples of E1 ubiquitin-activating enzymes include, for example, UBA1, UBA2, UBA3, UBA5, UBA6, UBA7, ATG7, NAE1, and SAE1.
E2泛素结合酶的例子包括，例如，hCdc34，Ubc-Uev1A、UBE2A、UBE2B、UBE2C、UBE2D1、UBE2D2、UBE2D3、UBE2D4、UBE2E1、UBE2E2、UBE2E3、UBE2F、UBE2G1、UBE2G2、UBE2H、UBE2I、UBE2J1、UBE2J2、UBE2K、UBE2L3、UBE2L6、UBE2M、UBE2N、UBE2O、UBE2Q1,UBE2Q2、UBE2R1(CDC34)、UBE2R2、UBE2S、UBE2T、UBE2U、UBE2V1、UBE2V2、UBE2W、UBE2Z、ATG3、BIRC6、和UFC1。Examples of E2 ubiquitin-conjugating enzymes include, e.g., hCdc34, Ubc-Uev1A, UBE2A, UBE2B, UBE2C, UBE2D1, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3, UBE2F, UBE2G1, UBE2G2, UBE2H, UBE2I, UBE2J1, UBE2J 2 , UBE2K, UBE2L3, UBE2L6, UBE2M, UBE2N, UBE2O, UBE2Q1, UBE2Q2, UBE2R1(CDC34), UBE2R2, UBE2S, UBE2T, UBE2U, UBE2V1, UBE2V2, UBE2W, UBE2Z, ATG3, BIRC6, and UFC1.
E3泛素连接酶的例子包括，例如，冯·希佩尔·林道(von Hippel–Lindau，VHL),Cereblon(CRBN),凋亡抑制蛋白(inhibitor of apoptosis protein,IAP),海螺状ECH相关蛋白1(Kelch-like ECH-associated protein 1,Keap1),RNF4,RNF114,MDM2、LUBAC,FBW7,Met30,HECT、SKP2、beta TRCP1，HUWEI，TRAF6，SMURF1,和E6AP。在某些实施方案中，E3泛素连接酶的例子包括，例如，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、和MKRN1。Examples of 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. In certain embodiments, 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.
内体-溶酶体通路成员包括，例如AP-1、AP-2、AP-3、内体、溶酶体、HOPS、ESCRT、GASP、BLOC-1、ESCRT、Retromer、ESCRT、sortingnexin、Dapper2、SNX4、Pincher、Rap1-PDZ-GEF1、clathrin、C3G/CrkL/Shp2/Gab2等。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.
自噬降解通路成员包括，例如分子伴侣介导的自噬(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等。Members of the autophagy degradation pathway include, for example, chaperone-mediated autophagy (CMA), USP10, G3BP1, ULK1, ATG16L1, TRIM16, FBXO27VDAC, RHOT1, MFN1/2, BNIP3L, FUNDC1, BNIP3, AMBRA1, BCL2LI3, FKBP8, CHDH, DISC1, PHB2, Cardiolipin, SEC62, RTN3, PEX5, PEX14, ABCD3, NUFIP1, Hsc70, etc.
在一些实施方案中，所述蛋白降解通路成员结合结构域是VHL结合结构域。在一些实施方案中，所述VHL结合结构域包含如SEQ ID NO:163(DRHDS(p)GLDS(p)M)或如SEQ ID NO:164(ALAPYIP)的氨基酸序列。In some embodiments, the protein degradation pathway member binding domain is a VHL binding domain. In some embodiments, the VHL binding domain comprises an amino acid sequence such as SEQ ID NO: 163 (DRHDS(p)GLDS(p)M) or such as SEQ ID NO: 164 (ALAPYIP).
在一些实施方案中，所述蛋白降解通路成员结合结构域是Keap1结合结构域。在一些实施方案中，所述Keap1结合结构域包含如SEQ ID NO:165(LDPETGEYL)的氨基酸序列。In some embodiments, the protein degradation pathway member binding domain is a Keap1 binding domain. In some embodiments, the Keap1 binding domain comprises an amino acid sequence such as SEQ ID NO: 165 (LDPETGEYL).
在一些实施方案中，所述蛋白降解通路成员结合结构域是E3泛素连接酶结合结构域，其包含如SEQ ID NO:166(DRHDSGLDSM)的氨基酸序列。In some embodiments, 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).
在一些实施方案中，所述蛋白降解通路成员结合结构域是CMA结合结构域。在一些实施方案中，所述CMA结合结构域包含如SEQ ID NO:167(KFERQ)的氨基酸序列。在一些实施方案中，所述CMA结合结构域包含如SEQ ID NO:168(KFERQKILDQRFFE)的氨基酸序列。In some embodiments, the protein degradation pathway member binding domain is a CMA binding domain. In some embodiments, the CMA binding domain comprises an amino acid sequence such as SEQ ID NO: 167 (KFERQ). In some embodiments, the CMA binding domain comprises an amino acid sequence such as SEQ ID NO: 168 (KFERQKILDQRFFE).
在一些实施方案中，所述蛋白降解通路成员结合结构域是蛋白酶体结合结构域。在一些实施方案中，所述蛋白酶体结合结构域选自：酵母Rad23(例如，S.cerevisiae Rad23)、人Rad23b(hHR23b)的类泛素(ubiquitin-like,UbL)结构域、HPV E7、锚蛋白的蛋白酶体结合结构域。在一些实施方案中，所述蛋白酶体结合结构域包含酵母Rad23的第1-77位氨基酸。在一些实施方案中，所述蛋白酶体结合结构域包含人Rad23b的第1-83位氨基酸。In some embodiments, the protein degradation pathway member binding domain is a proteasome binding domain. In some embodiments, the proteasome binding domain is selected from the group consisting of: yeast Rad23 (e.g., S. cerevisiae Rad23), ubiquitin-like (UbL) domain of human Rad23b (hHR23b), HPV E7, anchor Proteasome binding domain of the protein. In some embodiments, the proteasome binding domain comprises amino acids 1-77 of yeast Rad23. In some embodiments, the proteasome binding domain comprises amino acids 1-83 of human Rad23b.
本申请发明人发现，相比基础的TPD设计(Targeted protein binding domain-Transmembrane domain-ER retention domain，TBD-TMD-ERD)，包含蛋白降解通路成员结合结构域(ligand for E3 Ligase(E3L)，ligand for E2 Ubiquitin-conjugating enzyme(E2L)或ligand for lysosome(LL))(例如，如上所述的那些)的嵌合蛋白构建体对靶蛋白的降解效果具有显著提升。来自病毒ER驻留蛋白的功能结构和ERAD逆转运器复合物的结合能力不同，天然结构与ER驻留的E3ligase相互作用也存在差异性，同时还要和内质网内正常的错误折叠蛋白ERAD机制降解途径竞争，所以，天然的TMD-ERD的结构域对靶蛋白的泛素化和降解能力存在差异性，为了进一步提高靶蛋白的降解 效率，引入泛素-蛋白酶体系统(UPS)配体结构域和/或自噬-溶酶体途径(ALP)配体结构域能进一步把滞留在内质网的靶蛋白进行泛素化后实现蛋白酶体降解或通过溶酶体实现靶蛋白降解。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.
在一些实施方式中，所述嵌合蛋白构建体的靶向结构域或ERAD机制蛋白结合结构域通过铰链或连接子与蛋白降解通路成员结合结构域相连。在一些实施方式中，所述铰链包含如SEQ ID NO:147(IgG4铰链)或SEQ ID NO:144((Gly4Ser)2)所示的氨基酸序列。In some embodiments, the targeting domain or ERAD machinery protein binding domain of the chimeric protein construct is connected to the protein degradation pathway member binding domain through a hinge or linker. In some embodiments, the hinge comprises the amino acid sequence set forth in SEQ ID NO: 147 (IgG4 hinge) or SEQ ID NO: 144 ((Gly4Ser)2).
4.TPD嵌合蛋白构建体4.TPD chimeric protein construct
在一些实施方案中，本公开提供的嵌合蛋白构建体包含靶向TCR的靶向蛋白结合结构域和腺病毒E3-19K以及一个或多个上文所述的蛋白降解通路成员结合结构域。在一些实施方案中，本公开提供的嵌合蛋白构建体包含靶向TCR的靶向蛋白结合结构域和腺病毒E3-19K以及E3泛素连接酶结合结构域。在一些实施方案中，所述E3泛素连接酶结合结构域包含如SEQ ID NO:162(DRHDSGLDSMGSGSGALAPYIP)所示的氨基酸序列。In some embodiments, 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).
在一些实施方案中，本公开提供的嵌合蛋白构建体包含靶向TCR的靶向蛋白结合结构域和除了腺病毒E3-19K以外的病毒内质网驻留蛋白的跨膜结构域或其功能变体和内质网驻留结构域或其功能变体。所述病毒内质网驻留蛋白可以是选自下组的至少一种：HCMV糖蛋白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和牛痘病毒蛋白CPXV203。In some embodiments, 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.
在一些实施方案中，本公开提供的嵌合蛋白构建体包含靶向上述靶蛋白(例如，TCR)的靶向蛋白结合结构域和HCMV US2和/或US11的跨膜结构域或其功能变体和内质网驻留结构域或其功能变体。In some embodiments, 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.
在一些实施方案中，本公开提供的嵌合蛋白构建体包含靶向上述靶蛋白(例如，TCR)的靶向蛋白结合结构域和HCMV US3的跨膜结构域或其功能变体和内质网驻留结构域或其功能变体。In some embodiments, 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.
在一些实施方案中，本公开提供的嵌合蛋白构建体包含靶向上述靶蛋白(例如，TCR)的靶向蛋白结合结构域和HCMV US10的跨膜结构域或其功能变体和内质网驻留结构域或其功能变体。In some embodiments, 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.
在一些实施方案中，本公开提供的嵌合蛋白构建体包含靶向上述靶蛋白(例如，TCR)的靶向蛋白结合结构域和HCMV US6、HSV ICP47、CPXV012、EBV BNFL2a和/或BHV UL49.5的跨膜结构域或其功能变体和内质网驻留结构域或其功能变体。In some embodiments, 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. The transmembrane domain or functional variant thereof and the endoplasmic reticulum resident domain or functional variant thereof of 5.
在一些实施方案中，本公开提供的嵌合蛋白构建体包含靶向上述靶蛋白(例如，TCR)的靶向蛋白结合结构域和HHV-7US21的跨膜结构域或其功能变体和内质网驻留结构域或其功能变体。In some embodiments, 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.
在一些实施方案中，本公开提供的嵌合蛋白构建体包含靶向上述靶蛋白(例如，TCR)的靶向蛋白结合结构域和HCMV UL16,UL141和/或UL142的跨膜结构域或其功能变体和内质网驻留结构域或其功能变体。In some embodiments, 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.
在一些实施方案中，本公开提供的嵌合蛋白构建体包含靶向上述靶蛋白(例如，TCR)的靶向蛋白结合结构域和HIV Nef、HIV Vpu、HHV-7U21、HHV-8KK3、HHV-8KK5、MHV-68MK3和/或HTLV-1p12的跨膜结构域或其功能变体和内质网驻留结构域或其功能变体。In some embodiments, 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.
在一些实施方案中，本公开提供的嵌合蛋白构建体包含靶向上述靶蛋白(例如，TCR)的靶向蛋白结合结构域和Cowpox Virus蛋白CPXV203的跨膜结构域或其功能变体和内质网驻留结构域或其功能变体。In some embodiments, 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.
在一些实施方案中，本公开提供的嵌合蛋白构建体包含不是靶向TCR的靶向蛋白结合结构域和腺病毒E3-19K。In some embodiments, the present disclosure provides chimeric protein constructs comprising a targeting protein binding domain that is not TCR-targeting and adenovirus E3-19K.
在一些实施方案中，上述嵌合蛋白构建体进一步包括一个或多个上文所述的蛋白降解通路成员结合结构域。In some embodiments, the chimeric protein constructs described above further comprise one or more protein degradation pathway member binding domains as described above.
5.共表达部分5. Co-expression part
在一些实施方案中，本公开所提供的嵌合蛋白构建体和至少一个共表达部分共同表达。任何适用于共同表达的方式都可以使用。例如，嵌合蛋白构建体(或其中的一部分)和所述至少一个共表达部分可以通过可断裂的连接子连接，当连接子断裂后，则嵌合蛋白构建体和所述共表达部分可以共同表达。In some embodiments, 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. For example, 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.
在一些实施方案中，本公开所提供的嵌合蛋白构建体和至少一个共表达部分连接，例如通过可自我断裂的连接子(self-cleavable linkage)进行连接。在一些实施方案中，所述可自我断裂的连接子为可切割肽；例如，T2A肽、GSG-T2A肽、E2A肽、GSG-E2A肽、F2A肽、GSG-F2A肽、P2A肽或GSG-P2A肽。In some embodiments, 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. In some embodiments, 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.
在一些优选的实施方案中，所述T2A包含与SEQ ID NO.144所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述T2A的氨基酸序列如SEQ ID NO.144所示。In some preferred embodiments, 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.
在一些优选的实施方案中，GSG-T2A肽的氨基酸序列如SEQ ID NO.145所示。In some preferred embodiments, the amino acid sequence of the GSG-T2A peptide is as shown in SEQ ID NO. 145.
在一些优选的实施方案中，所述P2A包含与SEQ ID NO.146所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述P2A的氨基酸序列如SEQ ID NO.146所示。In some preferred embodiments, 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.
在一些优选的实施方案中，GSG-P2A肽的氨基酸序列如SEQ ID NO.147所示。In some preferred embodiments, the amino acid sequence of the GSG-P2A peptide is as shown in SEQ ID NO. 147.
在一些优选的实施方案中，所述E2A包含与SEQ ID NO.148所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述E2A的氨基酸序列如SEQ ID NO.148所示。In some preferred embodiments, 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.
在一些优选的实施方案中，GSG-E2A肽的氨基酸序列如SEQ ID NO.149所示。 In some preferred embodiments, the amino acid sequence of the GSG-E2A peptide is shown in SEQ ID NO. 149.
在一些优选的实施方案中，所述F2A包含与SEQ ID NO.150所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述F2A的氨基酸序列如SEQ ID NO.150所示。In some preferred embodiments, 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.
在一些优选的实施方案中，GSG-F2A肽的氨基酸序列如SEQ ID NO.151所示。In some preferred embodiments, the amino acid sequence of the GSG-F2A peptide is as shown in SEQ ID NO. 151.
本申请提供的所述共表达部分可以是任何具有生物功能的蛋白或者多肽。根据期望实现的生物学功能，可以选择合适的共表达部分。例如，为了降低细胞的免疫原性或抗原呈递，可以选择能够降解或减少MHC I类或II类分子的蛋白作为共表达部分。再例如，为了使细胞能够识别靶蛋白，可以选择识别或结合靶蛋白的结合结构域(例如趋化因子受体、或嵌合抗原受体CAR)作为共表达部分。再例如，为了增加免疫刺激活性，可以选择免疫刺激性分子作为共表达部分。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.
在一些优选的实施方案中，所述共表达部分的例子包括但不限于，完整的病毒ER驻留糖蛋白(例如，HCMV US2、US3、US11、US10、腺病毒E3-Kl 9、HCMV US6、HSV ICP47)、嵌合抗原受体(CAR)、功能性T细胞受体(TCR)、趋化因子受体(例如，CCR4、CCR5、CCR6、CCR7、CCR9、CCR2b、CXCR1、CXCR2和CXCR4)、NK细胞激活受体(例如，NKG2D、NKG2C、NKG2E、NKG2F、NKG2H、CD94、KIR2DL4、KIR2DS1、KIR2DS2、KIR2DS4、KIR3DS1，天然细胞毒性受体、TRAIL、DNAM-1、CD16a、2B4、NTB-A、CRACC以及NKp80)、CNK信号转接组件、细胞因子、CD7、免疫刺激性分子(例如，TNF-a、IFN-β、IFN-γ、IL-1、IL-2、IL-4、IL-6、IL-8、IL-10、IL-12、IL-18和粒细胞巨噬细胞集落刺激因子中的一种或多种)等。In some preferred embodiments, 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, CD7, immunostimulatory molecules (e.g., TNF-a, IFN-β, IFN-γ, IL-1, IL-2, IL-4, IL-6 , IL-8, IL-10, IL-12, IL-18 and one or more of granulocyte macrophage colony-stimulating factor), etc.
i)完整的病毒ER驻留糖蛋白i) Intact viral ER-resident glycoprotein
在一些实施方案中，所述共表达部分是完整的病毒ER驻留糖蛋白，包括但不限于，HCMV US2、US3、US11、US10、腺病毒E3-Kl 9、HCMV US6和HSV ICP47。In some embodiments, 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.
在一些优选的实施方案中，所述HCMV糖蛋白US2的全长序列包含与SEQ ID NO.82所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，优选地，具有降解MHC I类或II类分子的活性；所述HCMV糖蛋白US2的全长序列的氨基酸序列如SEQ ID NO.82所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述US2的全长序列包含与SEQ ID NO.110所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，优选地，具有降解MHC I类或II类分子的活性；所述US2的全长序列的氨基酸序列如SEQ ID NO.110所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HCMV糖蛋白US3的全长序列包含与SEQ ID NO.85所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，优选地，具有降解MHC I类或II类分子的活性；所述HCMV糖蛋白US3的全长序列的氨基酸序列如SEQ ID NO.85所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述US3的全长序列包含与SEQ ID NO.113所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，优选地，具有降解MHC I类或II类分子的活性；所述US3的全长序列的氨基酸序列如SEQ ID NO.113所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HCMV糖蛋白US11的全长序列包含与SEQ ID NO.88所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，优选地，具有降解MHC I类或II类分子的活性；所述HCMV糖蛋白US11的全长序列的氨基酸序列如SEQ ID NO.88所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述US11的全长序列包含与SEQ ID NO.116所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，优选地，具有降解MHC I类或II类分子的活性；所述US11的全长序列的氨基酸序列如SEQ ID NO.116所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HCMV糖蛋白US10的全长序列包含与SEQ ID NO.91所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，优选地，具有降解MHC I类或II类分子的活性；所述HCMV糖蛋白US10的全长序列的氨基酸序列如SEQ ID NO.91所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述腺病毒E3-19K的全长序列包含与SEQ ID NO.94所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，优选地，具有降解MHC I类或II类分子的活性；所述腺病毒E3-19K的全长序列的氨基酸序列如SEQ ID NO.94所示。In some preferred embodiments, 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.
在一些优选的实施方案中，所述HCMV US6的全长序列包含与SEQ ID NO.100所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，优选地，具有降解MHC I类或II类分子的活性；所述HCMV US6的全长序列的氨基酸序列如SEQ ID NO.100所示。 In some preferred embodiments, the full-length sequence of HCMV US6 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，所述HSV ICP47的全长序列包含与SEQ ID NO.102所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列，优选地，具有降解MHC I类或II类分子的活性；所述HSV ICP47的全长序列的氨基酸序列如SEQ ID NO.102所示。In some preferred embodiments, 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.
ii)嵌合抗原受体(CAR)ii)Chimeric Antigen Receptor (CAR)
在某些实施方式中，所述共表达部分是嵌合抗原受体(CAR)。所述CAR包含靶蛋白结合域(例如，靶向肿瘤的胞外识别结构域)、跨膜结构域和免疫受体活化信号传导结构域(ITAM)(也称为“胞内信号传导结构域”)。在某些实施方式中，所述CAR还可以进一步包含共刺激结构域。在某些实施方式中，所述靶向肿瘤的胞外识别结构域、跨膜结构域和/或胞内信号结构域之间包含铰链或接头。In certain embodiments, the co-expressed moiety is a chimeric antigen receptor (CAR). The 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" ). In certain embodiments, the CAR may further comprise a costimulatory domain. In certain embodiments, the tumor-targeting extracellular recognition domain, transmembrane domain and/or intracellular signaling domain includes a hinge or linker.
在一些实施方案中，所述靶向肿瘤的胞外识别结构域选自肿瘤抗原特异结合结构域、肿瘤微环境靶抗原结合结构域和/或靶向肿瘤微环境的趋化受体。In some embodiments, 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.
在一些优选的实施方案中，所述靶向肿瘤的胞外识别结构域选自能靶向识别肿瘤相关抗原的抗体或其功能片段、TCR或其组合；所述抗体的功能片段选自Fd、Fv、Fab、Fab’、F(ab’)2、Fv(scFv)、单链抗体(scFv)或纳米抗体(nanobody)、双链抗体、三链抗体和四链抗体。In some preferred embodiments, 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.
在一些优选的实施方案中，本文所述的CAR的跨膜结构域可来源于任何膜结合蛋白或跨膜蛋白，包括但不限于BAFFR、BLAME(SLAMF8)、CD2、CD3ε、CD4、CD5、CD8、CD9、CD11a(CD18、ITGAL、LFA-l)、CD11b、CD11c、CD11d、CD16、CD19、CD22、CD27、CD28、CD29、CD33、CD37、CD40、CD45、CD49a、CD49d、CD49f、CD64、CD80、CD84、CD86、CD96(Tactile)、CD100(SEMA4D)、CD103、CD134、CD137(4-1BB)、CD150(IPO-3、SLAMF1、SLAM)、CD154、CD160(BY55)、CD162(SELPLG)、CD226(DNAM1)、CD229(Ly9)、CD244(2B4、SLAMF4)、CD278(ICOS)、CEACAM1、CRT AM、GITR、HYEM(LIGHTR)、IA4、IL2Rβ、IL2Rγ、IL7R a、ITGA1、ITGA4、ITGA6、ITGAD、ITGAE、ITGAM、ITGAX、ITGB1、ITGB2、ITGB7、KIR、LTBR、OX40、NKG2C、NKG2D、NKp30、NKp44、NKp46、NKp80(KLRF1)、PAG/Cbp、PSGL1、SLAMF6(NTB-A、Ly108)、SLAMF7、T细胞受体的α、β或ζ链、TNFR2、VLA1和VLA-6。In some preferred embodiments, 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, CD3ε, CD4, CD5, CD8 , CD9, CD11a (CD18, ITGAL, LFA-l), CD11b, CD11c, CD11d, CD16, CD19, CD22, CD27, CD28, CD29, CD33, CD37, CD40, CD45, CD49a, CD49d, CD49f, CD64, CD80, CD84, CD86, CD96(Tactile), CD100(SEMA4D), CD103, CD134, CD137(4-1BB), CD150(IPO-3, SLAMF1, SLAM), CD154, CD160(BY55), CD162(SELPLG), CD226( DNAM1), CD229(Ly9), CD244(2B4, SLAMF4), CD278(ICOS), CEACAM1, CRT AM, GITR, HYEM(LIGHTR), IA4, IL2Rβ, IL2Rγ, IL7R a, ITGA1, ITGA4, ITGA6, ITGAD, ITGAE , ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIR, LTBR, OX40, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), PAG/Cbp, PSGL1, SLAMF6(NTB-A, Ly108), SLAMF7, T α, β or ζ chains of cellular receptors, TNFR2, VLA1 and VLA-6.
在一些优选的实施方案中，所述CAR的跨膜结构域选自NK细胞激活受体跨膜结构域、DAP10跨膜结构域、DAP12跨膜结构域、CD8跨膜结构域、CD28跨膜结构域、CD4跨膜结构域、4-1BB跨膜结构域、OX40跨膜结构域、ICOS跨膜结构域、CTLA-4跨膜结构域、PD-1跨膜结构域、LAG-3跨膜结构域、2B4跨膜结构域和BTLA跨膜结构域以及其组合。In some preferred embodiments, 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.
在一些优选的实施方案中，所述CAR的跨膜结构域选自CD8跨膜结构域、T细胞受体的α和/或β链跨膜结构域、CD28跨膜结构域、CD3ε跨膜结构域、CD45跨膜结构域、CD4跨膜结构域、CD5跨膜结构域、CD8跨膜结构域、CD9跨膜结构域、CD16跨膜结构域、CD22跨膜结构域、CD33跨膜结构域、CD37跨膜结构域、CD64跨膜结构域、CD80跨膜结构域、CD86跨膜结构域、CD134跨膜结构域、CD137跨膜结构域、CD154跨膜结构域、GITR跨膜结构域以及其组合。In some preferred embodiments, the transmembrane domain of the CAR is selected from the group consisting of CD8 transmembrane domain, α and/or β 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 .
在一些优选的实施方案中，所述免疫受体活化信号传导结构域(ITAM)来自免疫受体的胞内激活信号传导结构域；优选地，所免疫受体选自TCRζ、CD2、CD3γ、CD3δ、CD3ε、CD3ζ、CD5、CD22、FcRγ、CD66d、FcαRI、FcγRI、FcγRII、FcγRIII、Dectin-1、CLEC-1、CD72、CD79A、CD79B；优选地，所述免疫受体活化信号传导结构域(ITAM)与NK细胞信号转换器或其功能变体融合；优选地，所述免疫受体是CD3ζ。In some preferred embodiments, the immune receptor activation signaling domain (ITAM) is from an intracellular activation signaling domain of an immune receptor; preferably, the immune receptor is selected from TCRζ, CD2, CD3γ, CD3δ , CD3ε, CD3ζ, CD5, CD22, FcRγ, CD66d, FcαRI, FcγRI, FcγRII, FcγRIII, Dectin-1, CLEC-1, CD72, CD79A, CD79B; Preferably, the immune receptor activation signaling domain (ITAM ) is fused to an NK cell signal transducer or a functional variant thereof; preferably, the immune receptor is CD3ζ.
在一些优选的实施方案中，所述免疫受体活化信号传导结构域(ITAM)衍生自CD3ζ、共同FcRγ(FCER1G)、FcγRIIa、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD278(“ICOS”)、FcεRI CD66d、DAP10和DAP12等胞内信号结构域。In some preferred embodiments, the immunoreceptor activation signaling domain (ITAM) is derived from CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI CD66d, DAP10 and DAP12 and other intracellular signaling domains.
在一些优选的实施方案中，所述CAR的胞内信号传导结构域包括NK细胞激活受体的胞内信号结构域和/或共刺激信号传导结构域。In some preferred embodiments, the intracellular signaling domain of the CAR includes the intracellular signaling domain and/or the costimulatory signaling domain of an NK cell activating receptor.
在一些优选的实施方案中，所述T细胞共刺激信号传导结构域来自共刺激分子的胞内信号结构域；优选地，所述共刺激分子选自MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白质、细胞因子受体、整联蛋白、淋巴细胞活化信号分子(SLAM蛋白)、活化NK细胞受体、BTLA、Toll配体受体、OX40、CD2、CD7、CD16、CD27、CD28、CD30、CD40、CD38、CD35、CD79A、CD79B、CDS、ICAM-1、LFA-1、(CD11a/CD18)、4-1BB(CD137)、B7-H3、CDS、ICAM-1、ICOS(CD278)、GITR、BAFFR、LIGHT、HVEM(LIGHTR)、KIRDS2、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、NKG2D、NKG2C、NCR、DAP10、DAP12、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD19a、与CD83特异性结合的配体、CARD11、FcRa、FcRp、FcRy、Fyn、HVEM、ICOS、Lck、LAG3、LAT、LRP、NOTCH1、Wnt、OX40、ROR2、Ryk、SLAMF1、Slp76、pTa、 TCRa、TCRp、TRIM、ZAP70、PTCH2。更优选地，所述共刺激信号传导结构域可以选自NKG2D胞内信号结构域、DAP10胞内信号结构域、DAP12胞内信号结构域、NCR胞内信号结构域、CD28胞内信号结构域、4-1BB胞内信号结构域、OX40胞内信号结构域、ICOS胞内信号结构域。In some preferred embodiments, 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, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, NCR, DAP10, DAP12, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100SEMA4D ), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, Ligands that specifically bind to CD83, CARD11, FcRa, FcRp, FcRy, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, Wnt, OX40, ROR2, Ryk, SLAMF1, Slp76, pTa, TCRa, TCRp, TRIM, ZAP70, PTCH2. More preferably, the costimulatory signaling domain can be selected from the group consisting of NKG2D intracellular signaling domain, DAP10 intracellular signaling domain, DAP12 intracellular signaling domain, NCR intracellular signaling domain, CD28 intracellular signaling domain, 4-1BB intracellular signaling domain, OX40 intracellular signaling domain, and ICOS intracellular signaling domain.
在一些实施方案中，本文所述的CAR的铰链和/或跨膜域提供了CAR的胞外域的细胞表面呈现。本文所述的CAR的铰链可来源于任何膜结合蛋白或跨膜蛋白，包括但不限于BAFFR、BLAME(SLAMF8)、CD2、CD3ε、CD4、CD5、CD8、CD9、CD11a(CD18、ITGAL、LFA-l)、CD11b、CD11c、CD11d、CD16、CD19、CD22、CD27、CD28、CD29、CD33、CD37、CD40、CD45、CD49a、CD49d、CD49f、CD64、CD80、CD84、CD86、CD96(Tactile)、CD100(SEMA4D)、CD103、CD134、CD137(4-1BB)、CD150(IPO-3、SLAMF1、SLAM)、CD154、CD160(BY55)、CD162(SELPLG)、CD226(DNAM1)、CD229(Ly9)、CD244(2B4、SLAMF4)、CD278(ICOS)、CEACAM1、CRT AM、GITR、HYEM(LIGHTR)、IA4、IL2Rβ、IL2Rγ、IL7Ra、ITGA1、ITGA4、ITGA6、ITGAD、ITGAE、ITGAM、ITGAX、ITGB1、ITGB2、ITGB7、KIR、LTBR、OX40、NKG2C、NKG2D、NKp30、NKp44、NKp46、NKp80(KLRF1)、PAG/Cbp、PSGL1、SLAMF6(NTB-A、Ly108)、SLAMF7、T细胞受体的α、β或ζ链、TNFR2、VLA1和VLA-6。在一些实施方案中，本文所述的CAR的铰链包含CD8α的铰链区、人类免疫球蛋白(Ig)的铰链区或富含甘氨酸-丝氨酸的序列。In some embodiments, 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, CD3ε, CD4, CD5, CD8, CD9, CD11a (CD18, ITGAL, LFA- l), CD11b, CD11c, CD11d, CD16, CD19, CD22, CD27, CD28, CD29, CD33, CD37, CD40, CD45, CD49a, CD49d, CD49f, CD64, CD80, CD84, CD86, CD96(Tactile), CD100( SEMA4D), CD103, CD134, CD137(4-1BB), CD150(IPO-3, SLAMF1, SLAM), CD154, CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(Ly9), CD244(2B4 , SLAMF4), CD278(ICOS), CEACAM1, CRT AM, GITR, HYEM(LIGHTR), IA4, IL2Rβ, IL2Rγ, IL7Ra, ITGA1, ITGA4, ITGA6, ITGAD, ITGAE, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIR , LTBR, OX40, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG/Cbp, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, α, β or ζ chain of T cell receptor, TNFR2 , VLA1 and VLA-6. In some embodiments, the hinge of a CAR described herein comprises the hinge region of CD8α, the hinge region of human immunoglobulin (Ig), or a glycine-serine rich sequence.
在一些优选的实施方案中，所述接头是柔性接头；优选地，所述柔性接头包含所示的氨基酸序列(Gly(x)Ser(y))n，其中n是1到10的整数，并且x和y独立地是0到10的整数，前提是x和y不都是0；更优选地，所述接头包含SEQ ID NO.140所示的氨基酸序列或SEQ ID NO.141所示的氨基酸序列。In some preferred embodiments, the linker is a flexible linker; preferably, the flexible linker comprises the amino acid sequence shown (Gly(x)Ser(y))n, 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.
在一些优选的实施方案中，所述铰链是IgG1铰链或IgG4铰链。In some preferred embodiments, the hinge is an IgG1 hinge or an IgG4 hinge.
在一些优选的实施方案中，所述IgG1铰链包含与SEQ ID NO.142所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述IgG1铰链的氨基酸序列如SEQ ID NO.142所示。In some preferred embodiments, the IgG1 hinge comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，所述IgG4铰链包含与SEQ ID NO.143所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述IgG4铰链的氨基酸序列如SEQ ID NO.143所示。In some preferred embodiments, the IgG4 hinge comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
iii)CNK受体iii) CNK receptor
在某些实施方式中，所述共表达部分是CNK受体。所述CNK受体包含嵌合型NK激活受体组件和嵌合型NK信号转接组件(也称为“CNK信号转接组件”)。所述嵌合型NK激活受体组件至少可以包含NK细胞激活受体或其功能变体。In certain embodiments, 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.
NK细胞激活受体NK cell activating receptor
在一些实施方案中，所述NK细胞激活受体包含：(a)NK细胞激活受体胞外结构域(ED)或其功能变体，(b)NK细胞激活受体跨膜结构域(TMD)或其功能变体，以及(c)NK细胞激活受体胞内结构域(ICD)或其功能变体；任选地，所述NK细胞激活受体胞外结构域或其功能变体、所述NK细胞激活受体跨膜结构域或其功能变体和/或所述NK细胞激活受体胞内结构域或其功能变体之间包含铰链或接头。关于NK细胞激活受体和CNK信号转接组件的详细描述可参见，例如US 2020/0308248Al，其全部内容以引用的方式并入本文中。In some embodiments, the NK cell activating receptor comprises: (a) an NK cell activating receptor extracellular domain (ED) or a functional variant thereof, (b) an NK cell activating receptor transmembrane domain (TMD) ) or a functional variant thereof, and (c) an NK cell activating receptor intracellular domain (ICD) or a functional variant thereof; optionally, 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. A detailed description of NK cell activating receptors and CNK signal transduction components can be found, for example, in US 2020/0308248 Al, the entire content of which is incorporated herein by reference.
所述NK细胞激活受体选自NKG2D、NKG2C、NKG2E、NKG2F、NKG2H、CD94、KIR2DL4、KIR2DS1、KIR2DS2、KIR2DS4、KIR3DS1，天然细胞毒性受体(Natural Cytotoxicity Receptors，NCR)、TRAIL、DNAM-1、信号淋巴激活分子(signaling lymphocytic activation molecule，SLAM)家族分子2B4(又名CD244)、DNAX附件分子1(DNAM-1，又名CD226)、CD16a、2B4、NTB-A、CRACC(CS1)以及NKp80；其中，所述天然细胞毒性受体包括NKp46(又名NCR1或CD335)、NKp44(又名NCR2或CD336)和NKp30(又名NCR3或CD337)。在一些实施方案中，所述天然细胞毒性受体选自NKp46、NKp44和NKp30。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; Among them, 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). In some embodiments, the natural cytotoxic receptor is selected from NKp46, NKp44, and NKp30.
在一些优选的实施方案中，所述NK细胞激活受体是哺乳动物来源的NK细胞激活受体；优选地，所述哺乳动物选自人、灵长类动物、鼠、马、牛、绵羊、山羊、猫、猪、狗、美洲驼、羊驼、大象、松鼠、豚鼠。In some preferred embodiments, 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.
在一些优选的实施方案中，所述NK细胞激活受体是包含不同来源NK细胞激活受体结构域的重组NK细胞激活受体。In some preferred embodiments, the NK cell activating receptor is a recombinant NK cell activating receptor comprising NK cell activating receptor domains from different sources.
在一些优选的实施方案中，所述NK细胞激活受体是人源的NK细胞激活受体；优选地，所述NK细胞激活受体是包含不同的人源NK细胞激活受体结构域的重组NK细胞激活受体。In some preferred embodiments, the NK cell activating receptor is a human NK cell activating receptor; preferably, the NK cell activating receptor is a recombinant comprising different human NK cell activating receptor domains. NK cell activating receptor.
在一些优选的实施方案中，所述NK细胞激活受体是鼠源的NK细胞激活受体；优选地，所述NK细胞激活受体是包含不同的鼠源NK细胞激活受体结构域的重组NK细胞激活受体。In some preferred embodiments, the NK cell activating receptor is a murine NK cell activating receptor; preferably, the NK cell activating receptor is a recombinant comprising different murine NK cell activating receptor domains. NK cell activating receptor.
在一些优选的实施方案中，所述NK细胞激活受体是包含人源和鼠源NK细胞激活受体结构域的重组NK细胞激活受体。In some preferred embodiments, the NK cell activating receptor is a recombinant NK cell activating receptor comprising human and murine NK cell activating receptor domains.
在一些优选的实施方案中，所述NK细胞激活受体的胞外结构域为人或鼠的NK细胞激活受体的胞外结构域。In some preferred embodiments, the extracellular domain of the NK cell activating receptor is the extracellular domain of a human or murine NK cell activating receptor.
在一些优选的实施方案中，所述NK细胞激活受体的跨膜结构域为人或鼠的NK细胞激活受体的跨膜结构域。In some preferred embodiments, the transmembrane domain of the NK cell activating receptor is that of a human or murine NK cell activating receptor.
在一些优选的实施方案中，所述NK细胞激活受体的胞内结构域为人或鼠的NK细胞激活受体的胞内结构域。In some preferred embodiments, the intracellular domain of the NK cell activating receptor is the intracellular domain of a human or murine NK cell activating receptor.
在一些优选的实施方案中，所述NK细胞激活受体功能变体选自NK细胞激活受体的突变体，野生型融合蛋白，或野生型与突变型的融合蛋白。 In some preferred embodiments, the functional variant of the NK cell activating receptor is selected from a mutant of the NK cell activating receptor, a wild-type fusion protein, or a fusion protein of a wild-type and a mutant type.
在一些优选的实施方案中，人NKG2D的胞外域包含与SEQ ID NO.37所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2D的胞外域的氨基酸序列如SEQ ID NO.37所示。In some preferred embodiments, the extracellular domain of human NKG2D comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人NKG2D的全长序列包含与SEQ ID NO.38所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2D的全长序列的氨基酸序列如SEQ ID NO.38所示。In some preferred embodiments, 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.
在一些优选的实施方案中，小鼠NKG2D的胞外域包含与SEQ ID NO.39所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；小鼠NKG2D的胞外域的氨基酸序列如SEQ ID NO.39所示。In some preferred embodiments, the extracellular domain of mouse NKG2D comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，小鼠NKG2D的全长序列包含与SEQ ID NO:40所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；小鼠NKG2D的全长序的氨基酸序列如SEQ ID NO.40所示。In some preferred embodiments, the full-length sequence of mouse NKG2D comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 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.
在一些优选的实施方案中，人鼠重组NKG2D的全长序列包含与SEQ ID NO:41所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人鼠重组NKG2D的全长序列的氨基酸序列如SEQ ID NO.41所示。In some preferred embodiments, the full-length sequence of human mouse recombinant NKG2D comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 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.
在一些优选的实施方案中，人NKG2C的全长序列包含与SEQ ID NO.42所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2C的全长序列的氨基酸序列如SEQ ID NO.42所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人NKG2E的全长序列包含与SEQ ID NO：43所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2E的全长序列的氨基酸序列如SEQ ID NO.43所示。In some preferred embodiments, the full-length sequence of human NKG2E comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 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.
在一些优选的实施方案中，人NKG2F的全长序列包含与SEQ ID NO：44所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKG2F的全长序列的氨基酸序列如SEQ ID NO.44所示。In some preferred embodiments, the full-length sequence of human NKG2F comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 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.
在一些优选的实施方案中，人CD94的全长序列包含与SEQ ID NO.45所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；的氨基酸序列如SEQ ID NO.45所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人KIR2DL4的全长序列包含与SEQ ID NO：46所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR2DL4的全长序列的氨基酸序列如SEQ ID NO.46所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人KIR2DS1的全长序列包含与SEQ ID NO：47所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR2DS1的全长序列的氨基酸序列如SEQ ID NO.47所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人KIR2DS2的全长序列包含与SEQ ID NO：48所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR2DS2的全长序列的氨基酸序列如SEQ ID NO.48所示。In some preferred embodiments, the full-length sequence of human KIR2DS2 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 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.
在一些优选的实施方案中，人KIR2DS4的全长序列包含与SEQ ID NO：49所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR2DS4的全长序列的氨基酸序列如SEQ ID NO.49所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人KIR3DS1的全长序列包含与SEQ ID NO：50所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人KIR3DS1的全长序列的氨基酸序列如SEQ ID NO.50所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人NKp46的全长序列包含与SEQ ID NO.51所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKp46的全长序列的氨基酸序列如SEQ ID NO.51所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人NKp44的全长序列包含与SEQ ID NO.52所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKp44的全长序列的氨基酸序列如SEQ ID NO.52所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人NKp30的全长序列包含与SEQ ID NO.53所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKp30的全长序列的氨基酸序列如SEQ ID NO.53所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人DNAM1的全长序列包含与SEQ ID NO：54所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DNAM1的全长序列的氨基酸序列如SEQ ID NO.54所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人TRAIL的全长序列包含与SEQ ID NO：55所示氨基酸序列具有80％或以上同 一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人TRAIL的全长序列的氨基酸序列如SEQ ID NO.55所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人CD16a的全长序列包含与SEQ ID NO：56所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人CD16a的全长序列的氨基酸序列如SEQ ID NO.56所示。In some preferred embodiments, the full-length sequence of human CD16a comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 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.
在一些优选的实施方案中，人2B4的全长序列包含与SEQ ID NO：57所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人2B4的全长序列的氨基酸序列如SEQ ID NO.57所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人NTB-A的全长序列包含与SEQ ID NO：58所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NTB-A的全长序列的氨基酸序列如SEQ ID NO.58所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人CRACC的全长序列包含与SEQ ID NO：59所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人CRACC的全长序列的氨基酸序列如SEQ ID NO.59所示。In some preferred embodiments, the full-length sequence of human CRACC comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 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.
在一些优选的实施方案中，人NKp80的全长序列包含与SEQ ID NO：60所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人NKp80的全长序列的氨基酸序列如SEQ ID NO.60所示。In some preferred embodiments, 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.
CNK信号转接组件CNK signal transfer component
在一些实施方案中，所述CNK信号转接组件至少包含(i)NK细胞信号转换器(adaptor)(例如，DAP10或DAP12)或其功能变体。在一些实施方案中，所述NK细胞信号转换器包含：(a)NK细胞信号转换器胞外结构域(ED)或其功能变体，(b)NK细胞信号转换器跨膜结构域(TMD)或其功能变体，以及(c)NK细胞信号转换器胞内结构域(ICD)或其功能变体；任选地，所述NK细胞信号转换器胞外结构域或其功能变体、所述NK细胞信号转换器跨膜结构域或其功能变体和/或所述NK细胞信号转换器胞内结构域或其功能变体之间包含铰链或接头。In some embodiments, the CNK signaling component comprises at least (i) an NK cell signal adapter (eg, DAP10 or DAP12) or a functional variant thereof. In some embodiments, 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.
在一些优选的实施方案中，所述NK细胞信号转换器是哺乳动物来源的NK细胞信号转换器；优选地，所述哺乳动物选自人、灵长类动物、鼠、马、牛、绵羊、山羊、猫、猪、狗、美洲驼、羊驼、大象、松鼠、豚鼠。In some preferred embodiments, 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.
在一些优选的实施方案中，所述NK细胞信号转换器是包含不同来源NK细胞信号转换器结构域的重组NK细胞信号转换器。In some preferred embodiments, the NK cell signal transducer is a recombinant NK cell signal transducer comprising NK cell signal transducer domains from different sources.
在一些优选的实施方案中，所述NK细胞信号转换器是人源的NK细胞信号转换器；优选地，所述NK细胞信号转换器是包含不同的人源NK细胞信号转换器结构域的重组NK细胞信号转换器。In some preferred embodiments, the NK cell signal transducer is a human NK cell signal transducer; preferably, the NK cell signal transducer is a recombinant comprising different human NK cell signal transducer domains. NK cell signal transducer.
在一些优选的实施方案中，所述NK细胞信号转换器是鼠源的NK细胞信号转换器；优选地，所述NK细胞信号转换器是包含不同的鼠源NK细胞信号转换器结构域的重组NK细胞信号转换器。In some preferred embodiments, the NK cell signal transducer is a murine NK cell signal transducer; preferably, the NK cell signal transducer is a recombinant comprising different murine NK cell signal transducer domains. NK cell signal transducer.
在一些优选的实施方案中，所述NK细胞信号转换器是包含人源和鼠源NK细胞信号转换器结构域的重组NK细胞信号转换器。In some preferred embodiments, the NK cell signal transducer is a recombinant NK cell signal transducer comprising human and murine NK cell signal transducer domains.
在一些优选的实施方案中，所述NK细胞信号转换器的胞外结构域为人或鼠的NK细胞信号转换器的胞外结构域。In some preferred embodiments, the extracellular domain of the NK cell signal transducer is the extracellular domain of a human or murine NK cell signal transducer.
在一些优选的实施方案中，所述NK细胞信号转换器的跨膜结构域为人或鼠的NK细胞信号转换器的跨膜结构域。In some preferred embodiments, the transmembrane domain of the NK cell signal transducer is a transmembrane domain of a human or murine NK cell signal transducer.
在一些优选的实施方案中，所述NK细胞信号转换器的胞内结构域为人或鼠的NK细胞信号转换器的胞内结构域；In some preferred embodiments, the intracellular domain of the NK cell signal transducer is an intracellular domain of a human or murine NK cell signal transducer;
所述CNK信号转接组件中的所述NK细胞信号转换器为DAP10或DAP12。The NK cell signal converter in the CNK signal switching component is DAP10 or DAP12.
在一些优选的实施方案中，所述CNK细胞信号转换器功能变体选自DAP10或DAP12的突变体，或DAP10和DAP12的融合蛋白，或野生型DAP10或DAP12与突变型DAP10或DAP12的融合蛋白。In some preferred embodiments, the CNK cell signal transducer functional variant is selected from a mutant of DAP10 or DAP12, or a fusion protein of DAP10 and DAP12, or a fusion protein of wild-type DAP10 or DAP12 and mutant DAP10 or DAP12 .
在一些优选的实施方案中，所述CNK信号转接组件还包含(ii)免疫受体活化信号传导结构域(ITAM)和/或(iii)T细胞共刺激信号传导结构域。In some preferred embodiments, the CNK signaling component further comprises (ii) an immunoreceptor activation signaling domain (ITAM) and/or (iii) a T cell costimulatory signaling domain.
在一些优选的实施方案中，所述NK细胞信号转换器或其功能变体、所述免疫受体活化信号传导结构域(ITAM)和/或所述T细胞共刺激信号传导结构域之间包含铰链或接头；优选地，所述NK细胞信号转换器或其功能变体与所述免疫受体活化信号传导结构域(ITAM)域融合。In some preferred embodiments, 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; Preferably, the NK cell signal transducer or functional variant thereof is fused to the immune receptor activation signaling domain (ITAM) domain.
在一些优选的实施方案中，所述免疫受体活化信号传导结构域(ITAM)来自免疫受体的胞内激活信号传导结构域；优选地，所免疫受体选自TCRζ、CD2、CD3γ、CD3δ、CD3ε、CD3ζ、CD5、CD22、FcRγ、CD66d、FcαRI、FcγRI、FcγRII、FcγRIII、Dectin-1、CLEC-1、CD72、CD79A、CD79B；优选地，所述免疫受体活化信号传导结构域(ITAM)与NK细胞信号转换器或其功能变体融合；优选地，所述免疫受体是CD3ζ。In some preferred embodiments, the immune receptor activation signaling domain (ITAM) is from an intracellular activation signaling domain of an immune receptor; preferably, the immune receptor is selected from TCRζ, CD2, CD3γ, CD3δ , CD3ε, CD3ζ, CD5, CD22, FcRγ, CD66d, FcαRI, FcγRI, FcγRII, FcγRIII, Dectin-1, CLEC-1, CD72, CD79A, CD79B; Preferably, the immune receptor activation signaling domain (ITAM ) is fused to an NK cell signal transducer or a functional variant thereof; preferably, the immune receptor is CD3ζ.
在一些优选的实施方案中，所述T细胞共刺激信号传导结构域来自共刺激分子的胞内信号结构域。In some preferred embodiments, the T cell costimulatory signaling domain is derived from the intracellular signaling domain of a costimulatory molecule.
在一些优选的实施方案中，人DAP10的全长序列包含与SEQ ID NO.61所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有 98％或99％以上同一性的氨基酸序列；人DAP10的全长序列的氨基酸序列如SEQ ID NO.61所示。In some preferred embodiments, the full-length sequence of human DAP10 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP10的全长序列包含与SEQ ID NO.62所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10的全长序列的氨基酸序列如SEQ ID NO.62所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人DAP10的跨膜结构域包含与SEQ ID NO.63所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10的跨膜结构域的氨基酸序列如SEQ ID NO.63所示。In some preferred embodiments, the transmembrane domain of human DAP10 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP12的全长序列包含与SEQ ID NO.64所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12的全长序列的氨基酸序列如SEQ ID NO.64所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人DAP12的跨膜结构域包含与SEQ ID NO.65所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12的跨膜结构域的氨基酸序列如SEQ ID NO.65所示。In some preferred embodiments, the transmembrane domain of human DAP12 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP10和人DAP12的跨膜结构域融合蛋白包含与SEQ ID NO.66所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10和人DAP12的跨膜结构域的氨基酸序列如SEQ ID NO.66所示。In some preferred embodiments, the transmembrane domain fusion protein of human DAP10 and human DAP12 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP10-DAP12融合蛋白序列包含与SEQ ID NO.67所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-DAP12融合蛋白序列的氨基酸序列如SEQ ID NO.67所示。In some preferred embodiments, the human DAP10-DAP12 fusion protein sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人CD3zeta胞内信号传导结构域序列包含与SEQ ID NO.68所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人CD3zeta胞内信号传导结构域序列的氨基酸序列如SEQ ID NO.68所示。In some preferred embodiments, 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.
在一些优选的实施方案中，人DAP10-CD3zeta序列包含与SEQ ID NO.69所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-CD3zeta序列的氨基酸序列如SEQ ID NO.69所示。In some preferred embodiments, the human DAP10-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP12-CD3zeta序列包含与SEQ ID NO.70所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-CD3zeta序列的氨基酸序列如SEQ ID NO.70所示。In some preferred embodiments, the human DAP12-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP10-DAP12-CD3zeta序列包含与SEQ ID NO.71所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-DAP12-CD3zeta序列的氨基酸序列如SEQ ID NO.71所示。In some preferred embodiments, the human DAP10-DAP12-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人41BB胞内信号传导结构域序列包含与SEQ ID NO.72所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人41BB胞内信号传导结构域序列的氨基酸序列如SEQ ID NO.72所示。In some preferred embodiments, the human 41BB intracellular signaling domain sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP10-41BB序列包含与SEQ ID NO.73所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-41BB序列的氨基酸序列如SEQ ID NO.73所示。In some preferred embodiments, the human DAP10-41BB sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP10-41BB-CD3zeta序列包含与SEQ ID NO.74所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-41BB-CD3zeta序列的氨基酸序列如SEQ ID NO.74所示。In some preferred embodiments, the human DAP10-41BB-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人CD28胞内信号传导结构域序列包含与SEQ ID NO.75所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人CD28胞内信号传导结构域序列的氨基酸序列如SEQ ID NO.75所示。In some preferred embodiments, the human CD28 intracellular signaling domain sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP10-CD28序列包含与SEQ ID NO.76所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP10-CD28序列的氨基酸序列如SEQ ID NO.76所示。In some preferred embodiments, the human DAP10-CD28 sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP10-CD28-CD3zeta序列包含与SEQ ID NO.77所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优 选具有98％或99％以上同一性的氨基酸序列；人DAP10-CD28-CD3zeta序列的氨基酸序列如SEQ ID NO.77所示。In some preferred embodiments, the human DAP10-CD28-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP12-41BB序列包含与SEQ ID NO.78所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-41BB序列的氨基酸序列如SEQ ID NO.78所示。In some preferred embodiments, the human DAP12-41BB sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP12-41BB-CD3zeta序列包含与SEQ ID NO.79所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-41BB-CD3zeta序列的氨基酸序列如SEQ ID NO.79所示。In some preferred embodiments, the human DAP12-41BB-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP12-CD28序列包含与SEQ ID NO.80所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-CD28序列的氨基酸序列如SEQ ID NO.80所示。In some preferred embodiments, the human DAP12-CD28 sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，人DAP12-CD28-CD3zeta序列包含与SEQ ID NO.81所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；人DAP12-CD28-CD3zeta序列的氨基酸序列如SEQ ID NO.81所示。In some preferred embodiments, the human DAP12-CD28-CD3zeta sequence comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 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.
在一些优选的实施方案中，CNK信号转接组件包含选自SEQ ID NO.61～81所示的氨基酸序列。In some preferred embodiments, the CNK signal transfer component comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 61-81.
在一些优选的实施方案中，所述嵌合蛋白构建体和NK细胞激活受体和CNK信号转接组件形成多功能复合体，其中所述多功能复合体包含如SEQ ID NO.152所示的氨基酸序列。在一些优选的实施方案中，所述多功能复合体包含与SEQ ID NO.152所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；所述多功能复合体的氨基酸序列包含如SEQ ID NO.152所示的TCR抗体单链抗体。In some preferred embodiments, 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. In some preferred embodiments, 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.
6.核酸分子6. Nucleic acid molecules
在另一方面，本公开还提供了一种嵌合核酸构建体(chimeric nucleic acid construct)，其编码本申请所述的任一嵌合蛋白构建体。In another aspect, the disclosure also provides a chimeric nucleic acid construct encoding any of the chimeric protein constructs described in the application.
所述核酸分子中可以包括脱氧核糖核酸(DNA)、核糖核酸(RNA)、苏糖核酸(TNA)、乙二醇核酸(GNA)、肽核酸(PNA)、锁核酸(LNA、包括具有β-D-核糖构型的LNA、具有α-L-核糖构型的α-LNA(LNA的非对映异构体)、具有2'-氨基官能化的2'-氨基-LNA和具有2'-氨基官能化的2'-氨基-α-LNA)、乙烯核酸(ENA)、环己烯基核酸(CeNA)和/或它们的嵌合体和/或组合。示例性DNA包括但不限于质粒DNA(pDNA))等。示例性RNA包括但不限于mRNA、环状RNA、ccRNA。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 β- LNA with D-ribose configuration, α-LNA with α-L-ribose configuration (diastereomer of LNA), 2'-amino-LNA with 2'-amino functionalization and 2'-amino-LNA with 2'- Amino-functionalized 2'-amino-α-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 RNA, ccRNA.
在一些优选的实施方案中，所述核酸分子是mRNA。mRNA是编码至少一种蛋白质的任何RNA，并且可以在体外、体内、在原位或离体产生编码的蛋白质。本领域技术人员将理解，除非另有说明，本申请中阐述的核酸序列可以在代表性DNA序列中列举“T”，但在该序列代表RNA(例如，mRNA)的情况下，“T”将用“U”代替。因此，本文通过特定序列识别号公开和识别的任何DNA也公开了与DNA互补的相应RNA(例如，mRNA)序列，其中DNA序列的每个“T”被“U”取代。In some preferred embodiments, 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. Those skilled in the art will understand that, unless otherwise stated, 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". Accordingly, 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".
在一些优选的实施方案中，所述核酸分子包含与SEQ ID NO.154所示的核苷酸序列有80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的核苷酸序列。In some preferred embodiments, 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.
在一些优选的实施方案中，所述核酸分子是体外合成的mRNA。在一些优选的实施方案中，所述体外合成的mRNA具有修饰，可选地，包括一种或多种选自下组的修饰：5'UTR、5'UTR、聚A尾巴、5'-加帽、以及在编码区具有一个或多个修饰的核苷酸。In some preferred embodiments, the nucleic acid molecule is mRNA synthesized in vitro. In some preferred embodiments, 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.
i)核酸分子(例如mRNA)的修饰i) Modification of nucleic acid molecules (e.g. mRNA)
天然存在的真核细胞mRNA分子可以含有稳定元件，包括但不限于在其5'-末端的未翻译区(UTR)(5'UTR)和/或在其3'-末端的UTR(3'UTR)，以及以及其他结构特征，例如5'-帽结构、3'-聚A尾巴。5'UTR和3'UTR通常都从基因组DNA转录，并且是早产mRNA(premature mRNA)的部分。成熟mRNA的特征结构(如5'-帽结构和3'-聚A尾巴通常在mRNA加工过程中添加到转录的(早产)mRNA上。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. Both 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”是指mRNA的直接位于起始密码子(即核糖体翻译的mRNA转录物的第一个密码子)上游(即5')的区域，它不编码多肽。当生成RNA转录子时，5'UTR可能包含一个启动子序列。这样的启动子序列是本领域已知的。"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. When generating RNA transcripts, the 5'UTR may contain a promoter sequence. Such promoter sequences are known in the art.
“3'UTR”是指mRNA的直接位于终止密码子(即，指示翻译终止的mRNA转录物的密码子)下游(即3')的区域，其不编码多肽。"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.
“聚A尾巴”是mRNA的下游区域，例如，3'UTR的直接下游(即3')，包含多个连续单磷酸腺苷。一个聚A尾可能含有10到300个单磷酸腺苷。例如，聚A尾可能包含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或300单磷酸腺苷。在一些实施方案中，聚A尾含有50至250个单磷酸腺苷。在相关的生物学环境中(例如，在细胞中或在 体内)，聚A尾的作用是保护mRNA免于酶促降解(例如在细胞质中的酶促降解)，并有助于转录终止和/或mRNA从细胞核中的输出和翻译。3'-聚A尾通常是一段添加到转录的mRNA的3'-末端的腺嘌呤核苷酸。在某些情况下，它可以包含多达约400个腺嘌呤核苷酸。3'-聚A尾的长度可以影响mRNA分子本身的稳定性。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. For example, 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. In some embodiments, the polyA tail contains 50 to 250 adenosine monophosphate. in a relevant biological context (e.g., in cells or in in vivo ), 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.
多核苷酸的5'-加帽可以在体外转录反应期间同时完成，可根据制造商的使用规程用以下化学RNA帽类似物生成5'-鸟苷帽结构：3'-O-Me-m7G(5')ppp(5')G[ARCA帽]、G(5')ppp(5')A、G(5')ppp(5')G、m7G(5')ppp(5')A、m7G(5')ppp(5')G(新英格兰生物实验室，马萨诸塞州伊普斯威奇)。修饰的RNA的5'-加帽可以在转录后使用牛痘病毒加帽酶完成，以产生“Cap 0”结构：m7G(5')ppp(5')G(新英格兰生物实验室，马萨诸塞州伊普斯威奇)。Cap 1结构可以使用牛痘病毒加帽酶和2'-0甲基转移酶生成：m7G(5')ppp(5')G-2'-O-甲基。Cap 2结构可以由Cap 1结构产生，然后使用2'-O甲基转移酶对5'-倒数第三个(the 5'-antepenultimate)核苷酸进行2'-O-甲基化。Cap 3结构可以由Cap 2结构产生，然后使用2'-O甲基转移酶对5'-倒数第四个(the 5'-preantepenultimate)核苷酸进行2'-O-甲基化。酶可以来源于重组来源。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.
包含至少一个上述稳定元件的mRNA分子能够显著提高其编码的蛋白的表达量，并且当使用两个或更多的上述稳定元件时，其在蛋白表达量上相较于使用的单一稳定元件的mRNA显示出协同作用。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.
在一些实施方案中，本公开所述的mRNA被除去一个或更多AU丰富的序列。AU丰富的序列也可以被成为AURES，是在3'UTR发现的去稳定性序列。In some embodiments, 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.
在一些实施方案中，本公开所述的mRNA还包含编码信号肽的开放阅读框(ORF)。所述信号肽可以包含蛋白质N末端的第15-60位氨基酸，其通常是跨分泌通路上的膜转运所必需的，因此，普遍控制大多数蛋白质进入真核生物和原核生物中的分泌途径。所述信号肽的长度可能为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个氨基酸。In some embodiments, 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.
“开放阅读框”是一段连续的DNA，以起始密码子(例如，甲硫氨酸(ATG))开始，以终止密码子(例如，TAA、TAG或TGA)结尾，并编码多肽。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.
来自异源基因的信号肽在本领域中是已知的，并且可以测试所需的特性和然后掺入本公开的核酸中。在一些实施方案中，信号肽可以包括以下序列之一：MDSKGSSQKGSRLLLLLVVSNLLLPQGVVG(SEQ ID NO:155)、MDWTWILFLVAAATRVHS(SEQ ID NO:156)、METPAQLLFLLLLWLPDTTG(SEQ ID NO:157)、MLGSNSGQRVVFTILLVAPAYS(SEQ ID NO:158)、MKCLLYLAFLFIGVNCA(SEQ ID NO:159)、MWLVSLAIVTACAGA(SEQ ID NO:160)和MFVFLVLLPLVSSQC(SEQ ID NO:161)。Signal peptides from heterologous genes are known in the art and can be tested for desired properties and then incorporated into the nucleic acids of the present disclosure. In some embodiments, the signal peptide can include one of the following sequences: 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).
本申请所提供的核酸分子可以通过固相技术合成、液相化学合成、酶促连接及其组合合成。合成后的核酸分子可经纯化和定量。具体可参见WO2021222304，其全部内容以引用的方式并入本文中。The 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.
7.载体7. Carrier
在另一方面，本公开还提供了一种载体，其包含如本公开所提供的核酸分子，所述核酸分子可操作地连接至至少一个多核苷酸调控元件(例如，启动子)以表达所述核酸分子编码的嵌合蛋白构建体。In another aspect, 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.
在一些实施方案中，所述载体选自质粒、纳米质粒、粘粒、病毒载体、小环、RNA载体或线性或圆形DNA(例如，转座子DNA)或RNA分子。In some embodiments, the vector is selected from plasmids, nanoplasmids, cosmids, viral vectors, minicircles, RNA vectors, or linear or circular DNA (eg, transposon DNA) or RNA molecules.
在一些实施方案中，所述病毒载体选自逆转录病毒、慢病毒载体、腺病毒、细小病毒(例如，腺伴随病毒)、腺相关病毒(AAV)载体、冠状病毒、负链RNA病毒诸如正粘病毒(例如，流感病毒)、弹状病毒(例如，狂犬病和水疱性口炎病毒)、副粘病毒(例如，麻疼和仙台)、正链RNA病毒诸如小RNA病毒和甲病毒和双链DNA病毒，所述双链DNA病毒包括腺病毒、疱疹病毒(例如，单纯疱疹病毒1和2型、愛泼斯坦-巴尔病毒、巨细胞病毒)和痘病毒(例如，牛痘病毒、鸡痘病毒和金丝雀痘病毒)、诺沃克病毒、披膜病毒、黄病毒、呼肠孤病毒、乳多泡病毒、嗜肝DNA病毒、杆状病毒和肝炎病毒、病毒样颗粒(VLP)。In some embodiments, 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 canarypox virus), norovirus, togavirus, flavivirus, reovirus, papillomavirus, hepadnavirus, baculovirus and hepatitis virus, virus-like particles (VLP).
在一些实施方案中，所述病毒载体为逆转录病毒载体。In some embodiments, the viral vector is a retroviral vector.
在一些实施方案中，所述逆转录病毒选自禽造白细胞组织增生-肉瘤、哺乳动物C-型、B-型病毒、D-型病毒、HTLV-BLV集合、慢病毒、泡沫病毒。In some embodiments, 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.
在一些实施方案中，所述病毒载体为慢病毒载体。在一些实施方案中，所述病毒载体是溶瘤病毒载体。溶瘤病毒病毒载体是指基于溶瘤病毒的病毒载体，其中能够插入外源核酸序列。In some embodiments, 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.
在一些实施方案中，所述慢病毒载体选自HIV-1、HIV-2、SIV、FIV、BIV、EIAV、CAEV或绵羊脱髓鞘性脑白质炎慢病毒。In some embodiments, the lentiviral vector is selected from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or ovine demyelinating leukoencephalitis lentivirus.
在一些实施方案中，所述载体是基于转座子的表达载体。转座子是可以改变其在基因组内位置的DNA序列。在转座子系统中，编码本文所提供的嵌合蛋白构建体的核酸分子侧面是由介导转座子移动的转座酶可识别的末端重复序列。转座酶可以作为蛋白质共递送、与所述嵌合蛋白构建体编码在同一载体上或编码在单独的载体上。转座子系统的非限制性实例包括Sleeping Beauty、Piggyback、Frog Prince和Prince Charming。In some embodiments, the vector is a transposon-based expression vector. Transposons are DNA sequences that can change their position within the genome. In a transposon system, 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.
在一些实施方案中，本公开所提供的载体可以与其他载体组合，所述其他载体包含其他编码至少一个共表达部分的核酸分子。In some embodiments, vectors provided by the present disclosure can be combined with other vectors comprising other nucleic acid molecules encoding at least one co-expression moiety.
在一些优选的实施方案中，所述载体还包含启动子；优选地，所述启动子为EF1α启动子或CMV启动子。In some preferred embodiments, the vector further comprises a promoter; preferably, the promoter is an EF1α promoter or a CMV promoter.
在一些优选的实施方案中，本公开所提供的嵌合蛋白构建体可以与共表达部分在同一载体同一启动子，或不 同启动子下调控表达，或多种载体中进行表达。In some preferred embodiments, 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.
8.细胞8. Cells
在另一方面，本公开还提供了一种工程化细胞，其表达如本公开所提供的嵌合蛋白构建体，或包含如本公开所提供的嵌合核酸构建体或者载体。In another aspect, 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.
在一些实施方案中，所述工程化细胞同时表达如本公开所提供的嵌合蛋白构建体和上述所述的共表达部分。所述共表达部分和所述嵌合蛋白构建体的靶向蛋白结合结构域可以进行组合以实现特定目的。例如，当靶向蛋白结合结构域特异性识别免疫细胞上表达的能够引起免疫排异的蛋白时，所述共表达部分可以为进一步抑制免疫排异的蛋白(例如，病毒的ER驻留糖蛋白等)、靶向肿瘤微环境的蛋白(例如，趋化因子受体等)、刺激免疫活性的蛋白(例如，细胞因子、免疫细胞种系蛋白CD7、CD5等)，由此产生的细胞在移植到人体内时将具有降低的免疫原性(或免疫排斥)和/或具有增强的免疫活性。这对于细胞免疫治疗以及其他治疗性细胞移植中是非常有用的。因此，所述细胞可以是免疫细胞(例如，T细胞)、干细胞、肾细胞、胰岛细胞和心肌细胞等。In some embodiments, 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. For example, when the targeting protein binding domain specifically recognizes a protein expressed on immune cells that is capable of causing immune rejection, 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. Thus, the cells may be immune cells (eg, T cells), stem cells, kidney cells, pancreatic islet cells, cardiomyocytes, and the like.
在一些实施方案中，所述细胞是免疫细胞，其选自：T细胞、自然杀伤(NK)细胞、B细胞、巨噬细胞、单核细胞、树突细胞、中性粒细胞或γδT细胞。所述T细胞可以选自：CD8+T细胞、CD4+T细胞、细胞毒性T细胞、末端效应T细胞、记忆T细胞、幼稚T细胞、调节性T细胞组成的组细胞、自然杀伤T细胞、γ-δT细胞、细胞因子诱导的杀伤(CIK)T细胞和肿瘤浸润淋巴细胞。In some embodiments, the cells are immune cells selected from: T cells, natural killer (NK) cells, B cells, macrophages, monocytes, dendritic cells, neutrophils, or γδ 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, γ-δ T cells, cytokine-induced killer (CIK) T cells, and tumor-infiltrating lymphocytes.
在一些实施方案中，所述工程化细胞为T细胞。在表达上文所述的嵌合蛋白构建体和共表达部分以得到具有降低的免疫原性和/或增强的免疫活性的T细胞的情况下，所述T细胞还可以进一步表达能够特异性靶向肿瘤相关标记物的受体，包括但不限于CAR、TCR、NK细胞激活受体组件等。In some embodiments, the engineered cells are T cells. In the case where the chimeric protein constructs and co-expression portions described above are expressed to obtain T cells with reduced immunogenicity and/or enhanced immune activity, 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.
在一些实施方案中，所述工程化细胞为T细胞，其同时表达如本公开所提供的嵌合蛋白构建体和选自以下的一种或多种：腺病毒E3-K19、CAR、NK细胞激活受体和CNK信号转接组件。In some embodiments, 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.
在一些实施方案中，所述工程化细胞为T细胞，其同时表达如本公开所提供的嵌合蛋白构建体和NK细胞激活受体以及可选地CNK信号转接组件。In some embodiments, 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.
在一些实施方案中，所述工程化细胞为T细胞，其同时表达如本公开所提供的嵌合蛋白构建体、腺病毒E3-K19和CAR。In some embodiments, the engineered cells are T cells that simultaneously express a chimeric protein construct as provided by this disclosure, adenovirus E3-K19, and a CAR.
在另一方面，本公开还提供了一种产生本文所述的工程化细胞(例如，T细胞)的方法，其包括在适合表达如本文所述的核酸分子的条件下将如本文所述的载体引入起始细胞。In another aspect, 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.
本领域已知的许多产生CAR-T细胞的方法也可以应用于产生如本文所述的工程化细胞。例如，Zhang等人，Engineering CAR-T cells，Biomarker Research(2017)5:22中描述了产生CAR-T细胞的方法。本文提供的方法可以包括选自以下的一个或多个步骤：获得起始细胞、培养(包括扩增，任选地包括激活)起始细胞、和遗传修饰细胞。起始细胞可以是干细胞，所述干细胞可以是造血祖细胞(例如T细胞祖细胞、NK细胞祖细胞、巨噬细胞祖细胞)、造血干细胞(HSC)、CD34+细胞、胚胎细胞系干细胞、间充质干细胞或iPSC细胞。起始细胞也可以是从干细胞分化而来的细胞，例如上文所述的免疫细胞。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.
所述起始细胞可以从任何来源获得，例如，可以分离自受试者(例如，人类受试者)的免疫细胞(例如，T细胞)。在一些实施方案中，免疫细胞获自感兴趣的受试者，例如疑似患有特定疾病或病症的受试者、疑似患有特定疾病或病症的易感性的受试者、将经历、正在经历的受试者，或接受过针对特定疾病或病症的治疗，受试者也可以是健康志愿者或健康供体；免疫细胞也可以来自血库。所述免疫细胞对于感兴趣的受试者可以是自体的或同种异体的。所述免疫细胞可以从它们存在于受试者中的任何位置收集，包括但不限于血液、脐带血、脾脏、胸腺、淋巴结、胸腔积液、脾脏组织、肿瘤和骨髓。分离的免疫细胞可以直接使用，也可以保存一段时间，例如冷冻。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). In some embodiments, 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.
免疫细胞的激活和/或扩增是免疫细胞功能的主要步骤之一。在一些实施方案中，免疫细胞在遗传修饰的同时、之前或/和之后被激活和扩增。在一些实施方案中，免疫细胞在体外、离体或体内被激活和/或扩增。激活和扩增免疫细胞的方法已经在本领域中描述，并且可以用于本文描述的方法中。例如，T细胞可以通过与附接有刺激CD3/TCR复合体相关信号的药剂和刺激T细胞表面上的共刺激分子的配体的表面接触来激活和扩增。特别地，可以刺激T细胞群体，如通过与抗CD3抗体或其抗原结合片段或固定在表面上的抗CD2抗体接触，或与蛋白激酶C激活剂(例如苔藓虫素)以及钙离子载体接触。为了共刺激T细胞表面的辅助分子，使用与辅助分子结合的配体。例如，在适合刺激T细胞增殖的条件下，T细胞群可以与抗CD3抗体和抗CD28抗体接触。为了刺激CD4+T细胞或CD8+T细胞的增殖，可以使用抗CD3抗体和抗CD28抗体。在某些实施方案中，T细胞的初级刺激信号和共刺激信号可以由不同的方案提供。Activation and/or expansion of immune cells is one of the major steps in immune cell function. In some embodiments, immune cells are activated and expanded simultaneously with, before, or/and after genetic modification. In some embodiments, 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. For example, 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. In particular, 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. To costimulate accessory molecules on the surface of T cells, ligands that bind to the accessory molecules are used. For example, a population of T cells can be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. To stimulate the proliferation of CD4+ T cells or CD8+ T cells, anti-CD3 antibodies and anti-CD28 antibodies can be used. In certain embodiments, primary stimulatory signals and costimulatory signals for T cells can be provided by different protocols.
遗传修饰细胞可以通过用编码本文提供的嵌合蛋白构建体的核酸分子转导基本上同质的起始细胞来实现。在某些实施方案中，使用逆转录病毒载体(例如慢病毒载体)将本文提供的核酸分子引入起始细胞。例如，可以将本文提供的核酸分子克隆到慢病毒载体中，并且可以从其内源启动子、慢病毒长末端重复序列或对感兴趣的靶细胞类型特异的启动子驱动表达。用于递送病毒载体的常见递送方法包括但不限于电穿孔、显微注射、基因枪和磁转染。Genetically modifying cells can be accomplished by transducing substantially homogeneous starting cells with nucleic acid molecules encoding chimeric protein constructs provided herein. In certain embodiments, nucleic acid molecules provided herein are introduced into the starting cell using retroviral vectors (eg, lentiviral vectors). For example, 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.
遗传修饰细胞还可以通过用LNP递送核酸分子来实现，具体可参见上文“6.核酸分子”小节内容。其他用于核酸递送的非病毒方法包括使用磷酸钙、DEAE葡聚糖、电穿孔和原生质体融合进行体外转染。还可以使用转座酶或靶向核酸酶(例如锌指核酸酶、大范围核酸酶(meganucleases)或TALE核酸酶、CRISPR)来实现对起始细胞 的遗传修饰，从而获得如本公开所述的工程化细胞。Genetically modified cells can also be achieved by using LNP to deliver nucleic acid molecules. For details, please refer to the section "6. Nucleic acid molecules" above. Other non-viral methods for nucleic acid delivery include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Transposase or targeting nucleases (e.g. zinc finger nucleases, meganucleases or TALE nucleases, CRISPR) can also be used to achieve targeting of the starting cells. Genetic modification to obtain engineered cells as described in the present disclosure.
在某些实施方案中，本文提供的工程化细胞通过在施用之前将编码本文提供的嵌合蛋白构建体的核酸分子转染到起始细胞中来制备。在某些实施方案中，本文提供的工程化细胞可以通过用编码本文提供的嵌合蛋白构建体的核酸分子通过例如病毒载体转染免疫细胞来制备。本文提供的工程化细胞在细胞表面表现出降低的免疫原性分子(例如，TCR、HLA等)表达和/或免疫抑制分子(例如，PD-1等)表达和/或增强的肿瘤靶向性受体(例如CAR、工程化TCR、CNK受体等)表达。In certain embodiments, 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. In certain embodiments, 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.
在另一方面，本公开还提供了通过上述方法离体产生的细胞群。在某些实施方案中，至少70％、至少75％、至少80％、至少85％、至少90％或至少95％的细胞群表达可检测水平的本文提供的表达标记多肽(例如，与本文所述的嵌合蛋白构建体可切割连接的EGFR截短体(EGFRt))。EGFR截短体的表达水平可以代表本文所述的嵌合蛋白构建体在细胞群中的表达水平。In another aspect, the present disclosure also provides a population of cells produced ex vivo by the above method. In certain embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cell population express 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.
9.药物组合物、试剂盒9. Pharmaceutical compositions and kits
在另一方面，本公开还提供了一种药物组合物或一种试剂盒，其包含(i)如本文所述的嵌合蛋白构建体、核酸分子、载体或细胞群，以及(ii)药学上可接受的媒介。术语“药学上可接受的媒介”是指可促进本公开提供的嵌合蛋白构建体、核酸分子、表达载体和/或细胞群的储存和施用的任何和所有溶剂、分散介质、涂层、抗菌剂和抗真菌剂、等渗剂和吸收延迟剂等，药学上可接受的媒介可以包括任何合适的组分，例如但不限于盐水、脂质体、聚合赋形剂、胶体或载体颗粒。In another aspect, 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. The term "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. and antifungal agents, isotonic and absorption delaying agents, etc., and the pharmaceutically acceptable vehicle may include any suitable component, such as, but not limited to, saline, liposomes, polymeric excipients, colloids, or carrier particles.
在某些实施方案中，药学上可接受的媒介是可以溶解或分散本公开提供的嵌合蛋白构建体、核酸分子、表达载体和/或细胞群的盐水。盐水的说明性实例包括但不限于缓冲盐水、蔗糖溶液、生理盐水、醋酸盐缓冲液、磷酸盐缓冲液、柠檬酸盐缓冲液、碳酸氢盐缓冲液、盐溶液和聚山梨醇酯溶液。In certain embodiments, 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. Illustrative examples of 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.
i)核酸药物组合物i) Nucleic acid pharmaceutical composition
在某些实施方案中，药学上可接受的媒介是脂质体。脂质体是单层或多层囊泡，具有由亲脂性材料形成的膜和内部水性部分。本公开提供的核酸分子和/或载体可以封装在脂质体的水性部分中。示例性脂质体包括但不限于基于3[N-(N',N'-二甲基氨基乙烷)氨基甲酰基]胆固醇(DC-Chlo)的脂质体、基于N-(2,3-二油酰氧基)丙基-N,N,N-三甲基氯化铵(DOTMA)的脂质体，和基于1,2-二油酰氧基-3-三甲基丙烷(DOTAP)的脂质体。制备脂质体和将核酸分子和/或载体包封在脂质体中的方法在本领域中是众所周知的(参见，例如，D.D.Lasic et al,Liposomes in gene delivery,published by CRC Press,1997)。In certain embodiments, 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. Methods of preparing liposomes and encapsulating nucleic acid molecules and/or vectors in liposomes are well known in the art (see, e.g., D.D. Lasic et al, Liposomes in gene delivery, published by CRC Press, 1997) .
在某些实施方案中，药学上可接受的媒介是聚合赋形剂，包括但不限于微球、微胶囊、聚合胶束和树枝状聚合物。本公开提供的核酸分子和/或载体可以通过本领域已知的方法封装、粘附或包被在基于聚合物的组分上(参见，例如，W.Heiser,Nonviral gene transfer techniques,published by Humana Press,2004；U.S.patent 6025337；Advanced Drug Delivery Reviews,57(15):2177-2202(2005))。In certain embodiments, 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)).
在某些实施方案中，药学上可接受的媒介是胶体或载体颗粒，例如金胶体、金纳米颗粒、二氧化硅纳米颗粒和多段纳米棒。本公开提供的核酸分子和/或载体可以以本领域已知的任何合适的方式包被、粘附到载体或与载体结合(参见，例如，M.Sullivan et al.,Gene Therapy,10:1882–1890(2003),C.Mclntosh et al.,J.Am.Chem.Soc.,123(31):7626–7629(2001),D.Luo et al.,Nature Biotechnology,18:893-895(2000),and A.Salem et al.,Nature Materials,2:668-671(2003))。In certain embodiments, 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)).
在某些实施方案中，药物组合物可进一步包含添加剂，包括但不限于稳定剂、防腐剂和有助于药物的细胞摄取的转染促进剂。合适的稳定剂可以包括但不限于谷氨酸钠、甘氨酸、EDTA和白蛋白。合适的防腐剂可以包括但不限于2-苯氧乙醇、苯甲酸钠、山梨酸钾、羟基苯甲酸甲酯、酚类、硫柳汞和抗生素。合适的转染促进剂可以包括但不限于钙离子。In certain embodiments, pharmaceutical compositions may further include additives including, but not limited to, stabilizers, preservatives, and transfection enhancers that aid cellular uptake of the drug. Suitable stabilizers may include, but are not limited to, sodium glutamate, glycine, EDTA, and albumin. Suitable preservatives may include, but are not limited to, 2-phenoxyethanol, sodium benzoate, potassium sorbate, methyl hydroxybenzoate, phenols, thimerosal, and antibiotics. Suitable transfection promoters may include, but are not limited to, calcium ions.
本文提供的药物组合物可以通过本领域已知的任何合适的途径施用，包括但不限于，肠胃外、口服、肠内、颊、鼻、局部、直肠、阴道、肌内、鼻内、经粘膜、表皮、透皮、真皮、眼、肺和皮下给药途径。本文所提供的药物组合物可以以适合每种给药途径的制剂或制剂的形式施用于受试者。适用于药物组合物给药的制剂可以包括但不限于溶液、分散体、乳剂、粉剂、悬浮液、气雾剂、喷雾剂、滴鼻剂、基于脂质体的制剂、贴片、植入物和栓剂。Pharmaceutical 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.
制剂可以方便地以单位剂型存在并且可以通过药学领域中众所周知的任何方法制备。制备这些制剂或药物组合物的方法包括将本公开所述的核酸分子提供给一种或多种药学上可接受的媒介以及任选地一种或多种添加剂的步骤。制备此类制剂的方法可参见，例如，Remington's Pharmaceutical Sciences(Remington:The Science and Practice of Pharmacy,19th ed.,A.R.Gennaro(ed),Mack Publishing Co.,N.J.,1995；R.Stribling et al.,Proc.Natl.Acad.Sci.USA,89:11277-11281(1992)；A.Barnes et al.,Current Opinion in Molecular Therapeutics 2000 2:87-93(2000)；T.W.Kim et al.,The Journal of Gene Medicine,7(6):749-758(2005)；和S.F.Jia et al.,Clinical Cancer Research,9:3462(2003)；A.Shahiwala et al.,Recent patents on drug delivery and formulation,1:1-9(2007)；，其参考文献通过引用整体并入本文。The 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. Stribling et al., Proc.Natl.Acad.Sci.USA, 89:11277-11281(1992); A.Barnes et al., Current Opinion in Molecular Therapeutics 2000 2:87-93(2000); T.W.Kim et al., The Journal of Gene Medicine,7(6):749-758(2005); and S.F.Jia et al., Clinical Cancer Research,9:3462(2003); A.Shahiwala et al.,Recent patents on drug delivery and formulation,1: 1-9(2007);, the references of which are incorporated by reference in their entirety.
在一些实施方案中，所述核酸分子(例如mRNA)可以物理方法、生物方法或化学方法进行递送(参见，例如，S.Guan,J.Rosenecker,Gene Ther.2017,24,133.)。In some embodiments, the nucleic acid molecules (e.g., mRNA) can be delivered by physical, biological, or chemical methods (see, e.g., S. Guan, J. Rosenecker, Gene Ther. 2017, 24, 133.).
物理方法包括但不限于通过基因枪(例如用Au-颗粒的基因枪)、电穿孔、声穿孔等进行递送(参见，例如，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.)。 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.).
生物方法包括但不限于通过病毒载体(例如，逆转录病毒载体、腺病毒载体、腺相关病毒载体)进行递送。Biological methods include, but are not limited to, delivery via viral vectors (eg, retroviral vectors, adenoviral vectors, adeno-associated viral vectors).
化学方法包括但不限于通过天然蛋白/聚糖、聚合物、脂质递送。示例性天然蛋白/聚糖包括鱼精蛋白和壳聚糖(参见，例如，A.E.et al.,Cancer Immunol.Immunother.2015,64,1461；U.S.Kumar et al.ACS Nano 2021,11,17582)。示例性聚合物包括聚乙烯亚胺(PEI)(例如，线性PEI、分支PEI和树枝状PEI)、聚(β-氨基酯)(PBAE)(参见，例如，K.Singha et al.,Nucleic Acid Ther.2011,21,133；A.A.Eltoukhy et al.,Biomaterials 2012,33,3594)。Chemical methods include, but are not limited to, delivery via natural proteins/glycans, polymers, and lipids. Exemplary natural proteins/glycans include protamine and chitosan (see, e.g., AE 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).
示例性脂质包括阳离子脂质，例如1,2-diocta-decenyl-3-trimethy lammonium-propane(DOTMA)和1,2-dioleoyl-3-trimethylammo niumpropane(DOTAP)(参见，例如，X.Hou et al.,Nat.Rev.Mater.2021,10,1078.)、由DOTMA、DOTAP和DOPE(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine,辅助脂质)形成的脂质体，其可以与mRNA自组装后形成胶体稳定的纳米粒子(参见，例如，L.M.Kranz et al.,Nature 2016,534,396.)。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.).
示例性脂质还可以是可离子化脂质。可离子化脂质(pKa 6.5-6.9)是一种替代性脂质材料，在生理pH下呈中性，但在酸性环境中通过游离胺的质子化而带正电荷(参见，例如，S.C.Semple et al.,Nat.Biotechnol.2010,28,172)。细胞内化后，可离子化脂质形成的纳米颗粒被包裹在内体(endosome)中。随后，由于内体和溶酶体内pH值的不断下降，可离子化脂质获得质子进行离子化，从而促进脂质纳米颗粒(lipid nanoparticle，LNP)与内体膜的融合，最终使得装载在脂质纳米颗粒上的mRNA释放到细胞质中(参见，例如，L.Miao et al.,Mol.Cancer 2021,20,41.)。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. Subsequently, due to the continuous decrease of pH value in endosomes and lysosomes, 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. The mRNA on the plasma nanoparticles is released into the cytoplasm (see, for example, L. Miao et al., Mol. Cancer 2021, 20, 41.).
可离子化脂质可以与胆固醇、辅助脂质和聚乙二醇化脂质(即PEG化脂质)形成脂质纳米颗粒制剂。胆固醇是一种天然的刚性和疏水性脂质，可维持脂质纳米颗粒的结构和稳定性。它还可以促进装载mRNA的脂质纳米颗粒(即mRNA纳米颗粒)与内体膜的融合。辅助脂质(例如两性离子脂质DOPE、1,2-distearoyl-snglycero-3-phosphocholine(DSPC)和1,2-Dioleoyl-sn-glycero-3-phosphocholine(DOPC)被广泛用于促进细胞膜穿透和内体膜逃逸(参见，例如，N.Chaudhary et al.,Nat.Rev.Drug Discovery 2021,20,817.)。PEG化脂质由PEG和锚定脂质组成。亲水性PEG主要分布于mRNA复合体表面，而疏水区嵌入脂质双层内部。PEG化脂质的引入不仅增加了脂质纳米颗粒的半衰期也可以通过改变PEG链的分子量来调节粒径。通常，分子量和脂质尾长度的范围可以分别为350至3000Da和10至18个碳(参见，例如，N.Chaudhary et al.,Nat.Rev.Drug Discovery 2021,20,817.)。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. Generally, 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.).
在过去的几十年来，研究人员开发了用于mRNA递送的大型的可离子化脂质库，包括DLin-MC3-DMA、SM-102、TT3、C12-200、306Oi10和ALC-0315(参见，例如M.Yanez Arteta et al.,Proc.Natl.Acad.Sci.USA 2018,115,E3351；R.Verbeke et al.,Controlled Release 2021,333,511；B.Li et al.,Nano Lett.2015,15,8099；K.A.Hajj et al.,Nano Lett.2020,20,5167；K.A.Hajj et al.,Small 2019,15,1805097；A.B.Vogel et al.,Nature 2021,592,283)。其中一些在临床应用上取得了显著成果。一个典型的例子是DLin-MC3-DMA，其是经美国食品和药物管理局(FDA)批准用于siRNA递送的Onpattro的关键组分(参见，例如，A.Akinc et al.,Nat.Nanotechnol.2019,14,1084)。DLin-MC3-DMA也被广泛用于mRNA递送，包括蛋白质和肽替代、基因编辑和抗病毒感染(参见，例如，R.S.Riley et al.,Sci.Adv.2021,7,eaba1028.)。SM-102和ALC-0315这两个“明星分子”已被FDA批准分别为预防COVID-19的BNT162b和mRNA-1273疫苗中的关键成分(参见，例如，X.Hou et al.,Nat.Rev.Mater.2021,10,1078.)。Over the past few decades, researchers have developed a large library of ionizable lipids for mRNA delivery, including DLin-MC3-DMA, SM-102, TT3, C12-200, 306O i10 , and ALC-0315 (see , such as M.Yanez Arteta et al., Proc.Natl.Acad.Sci.USA 2018,115,E3351; R.Verbeke et al., Controlled Release 2021,333,511; B.Li et al., Nano Lett.2015, 15,8099; KAHajj et al., Nano Lett.2020, 20, 5167; KAHajj et al., Small 2019, 15, 1805097; ABVogel et al., Nature 2021, 592, 283). Some of them have achieved remarkable results in clinical applications. A typical example is DLin-MC3-DMA, which is a key component of Onpattro approved by the U.S. Food and Drug Administration (FDA) for siRNA delivery (see, e.g., A. Akinc et al., Nat. Nanotechnol. 2019,14,1084). 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.).
理想的基于脂质的mRNA载体必须满足以下条件：1)裸露的mRNA可以形成稳定的复合物，保护mRNA不被降解；2)应添加四个关键组件(可离子化脂质、胆固醇、辅助脂质和聚乙二醇化脂质)以稳定mRNA复合物；3)脂质纳米颗粒的成分应该被质子化以引发膜不稳定并促进mRNA复合物的内体逃逸；以及4)所有的脂质材料都是可生物降解且不会对患者造成任何伤害。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.
对基于脂质的递送平台进行优化时应当考虑以下几点：1)可离子化脂质的降解能力：脂质的骨架结构通过将炔基和酯基引入脂质尾部来促进脂质的清除并降低毒性；2)脂质纳米颗粒的免疫原性：脂质纳米粒内的杂环脂质可通过激活树突状细胞(DC)的干扰素基因刺激物(STING)通路来改善mRNA疫苗的效率(参见，例如，L.Miao et al.,Nat.Biotechnol.2019,37,1174.)；3)脂质纳米颗粒的稳定性：一些有潜力的策略有望提高mRNA的稳定性疫苗，包括pKa优化、赋形剂引入和mRNA修饰等。The following points should be considered when optimizing lipid-based delivery platforms: 1) Degradability of ionizable lipids: The backbone structure of lipids promotes lipid clearance and 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.
脂质纳米颗粒可以使用如本领域公知的组分、组合物和方法产生，参见例如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和PCT/US2016/069491，其全部内容均以引用方式并入本文。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.
ii)溶瘤病毒及其药物组合物ii) Oncolytic viruses and pharmaceutical compositions thereof
在一些实施方案中，本申请提供了一种能够表达本申请所述嵌合蛋白构建体的溶瘤病毒。在本申请中，“溶瘤病毒”是指能够通过感染肿瘤细胞，在肿瘤细胞中复制，并且溶解肿瘤细胞的任何病毒。在某些实施方式中，溶瘤病毒进一步能够在连续的复制循环中扩散至其他肿瘤细胞。In some embodiments, the present application provides an oncolytic virus capable of expressing a chimeric protein construct described herein. As used 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.
溶瘤病毒可源自多种病毒，其非限制性实例包括痘苗病毒、腺病毒、单纯疱疹病毒1(HSV1)、单纯疱疹病毒2(HSV2)、粘液瘤病毒、呼肠孤病毒、脊髓灰质炎病毒、水疱性口炎病毒(VSV)、麻疹病毒(MV)、拉沙病毒(LASV)和新城疫病毒(NDV)，以及这些病毒的变体。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.
在一些实施方案中，本申请提供了溶瘤病毒，其中包含如本申请所提供的核酸分子，所述核酸分子可操作地连接到至少一个多核苷酸调控元件(例如，启动子)以表达所述核酸分子编码的嵌合蛋白构建体。 In some embodiments, 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.
在一些实施方案中，本申请所述的溶瘤病毒可感染基本上任何细胞类型。在一些实施方案中，溶瘤病毒可以是复制选择性的。复制选择性的溶瘤病毒在肿瘤细胞里的复制比在非肿瘤细胞中的复制更多。In some embodiments, the oncolytic viruses described herein can infect essentially any cell type. In some embodiments, oncolytic viruses can be replication selective. Replication-selective oncolytic viruses replicate more in tumor cells than in non-tumor cells.
可以理解，如果病毒的复制处于基因表达调节因子的控制之下，例如源自白蛋白基因的5'侧的增强子/启动子区域，则可以使溶瘤病毒具有复制选择性(例如参见Miyatake等人，1997，J.Virol.71：5124-5132)。It will be appreciated that 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).
在一些实施方案中，溶瘤病毒在其基因组中可以具有肿瘤细胞特异性启动子，或肿瘤细胞特异性转录调控序列。“肿瘤细胞特异性”对于启动子或转录条款序列而言，是指其通常以高于正常细胞的水平存在于靶肿瘤细胞中。通过这种方式，可赋予溶瘤病毒增强水平的肿瘤细胞特异性。In some embodiments, an oncolytic virus may have a tumor cell-specific promoter, or tumor cell-specific transcriptional regulatory sequences in its genome. "Tumor 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.
例如，通过将HSV基因可操作地连接到TGF-β启动子，可以将HSV的主要转录单位置于肿瘤生长因子-β(TGF-β)启动子的转录控制之下。已知某些肿瘤细胞相对于相同类型的非肿瘤细胞过度表达TGF-β。因此，其中复制受到TGF-β启动子的转录控制的溶瘤病毒是复制选择性的，因为它在某些肿瘤细胞中比在相同类型的非肿瘤细胞中更能复制。类似的复制选择性溶瘤病毒可以使用任何已知可选择性地在受影响细胞中引起过表达的基因表达调节剂来制备。复制选择性溶瘤病毒可以是例如HSV-1突变体，其中编码ICP34.5的基因发生突变或缺失。溶瘤病毒还可以在其基因组中进一步包含其他修饰。例如，溶瘤病毒可以包含插入到UL44基因中的额外DNA。这种插入可以产生UL44基因的功能失活和产生的裂解表型，或者它可以插入到已经失活的基因中，或替代缺失的基因。For example, by operably linking the HSV gene to the TGF-β promoter, the major transcription unit of HSV can be placed under the transcriptional control of the tumor growth factor-β (TGF-β) promoter. Certain tumor cells are known to overexpress TGF-β relative to non-tumor cells of the same type. Therefore, an oncolytic virus in which replication is transcriptionally controlled by the TGF-β 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. For example, 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.
在一些实施方案中，溶瘤病毒被改造为将编码本申请所述嵌合蛋白构建体的核酸序列置于肿瘤细胞特异性启动子的控制下。In some embodiments, the oncolytic virus is engineered to place the nucleic acid sequence encoding the chimeric protein construct described herein under the control of a tumor cell-specific promoter.
在一些实施方案中，溶瘤病毒被改造为将至少一个病毒复制所需蛋白的编码基因置于肿瘤细胞特异性启动子的控制下。In some embodiments, the oncolytic virus is engineered to place a gene encoding at least one protein required for viral replication under the control of a tumor cell-specific promoter.
在一些实施方案中，所述溶瘤病毒载体可以表达本申请所述的嵌合蛋白构建体，所述嵌合蛋白构建体的靶向结构域能够识别并结合肿瘤蛋白。本申请前文所述的任意肿瘤相关蛋白或肿瘤相关靶点都适用。在某些实施方式中，肿瘤蛋白可以是在肿瘤细胞里高表达的蛋白(例如，c-Myc,Bcl-2、Bcl-xL、Bcl-w、KRAS)，在肿瘤细胞里表达的促进肿瘤细胞生长的蛋白(例如雌激素受体，雄激素受体，Her2、VEGF、VEGFR、PDGFRβ、EGFR、EGFR突变体)，或促进肿瘤细胞逃逸正常免疫反应的蛋白(例如PD-L1、TGF-β1)。In some embodiments, the oncolytic viral vector can express a chimeric protein construct described herein whose targeting domain is capable of recognizing and binding tumor proteins. Any tumor-related protein or tumor-related target described previously in this application is applicable. In certain embodiments, 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, PDGFRβ, EGFR, EGFR mutants), or proteins that promote tumor cells to escape normal immune responses (such as PD-L1, TGF-β1) .
在一些实施方案中，所述嵌合蛋白构建体的靶向结构域可以结合Bcl-2。任何能够靶向结合bcl-2的靶向结构域都可以使用，例如但不限于，开发针对Bcl-2抗凋亡家族成员蛋白高度保守和同源的BH1,BH2基序的纳米抗体序列可以作为靶向结构域，构建完成靶向Bcl-2家族蛋白的ER-TPD元件，并克隆装载在溶瘤病毒强启动子后，特异性的感染肿瘤细胞后，溶瘤病毒大量扩增，并表达Bcl-2家族蛋白的ER-TPD元件，实现细胞内Bcl-2抗凋亡成员蛋白的降解，直接导致肿瘤细胞凋亡。In some embodiments, the targeting domain of the chimeric protein construct can bind Bcl-2. Any targeting domain capable of binding bcl-2 can be used. For example, but not limited to, developing Nanobody sequences targeting the highly conserved and homologous BH1, BH2 motifs of Bcl-2 anti-apoptotic family member proteins can be used as Targeting domain, 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.
在一些实施方案中，所述嵌合蛋白构建体的靶向结构域可以结合VEGFR。任何能够靶向结合VEGFR的靶向结构域都可以使用，例如但不限于，VEGFR抗体(anti-VEGF-A抗体Bevacizumab and anti-VEGFR2抗体Ramucizumab,)的重链和轻链重组的scFv串联ER-TPD元件，构建靶向VEGFR ER-TPD元件，并克隆装载在溶瘤病毒强启动子后，特异性的感染肿瘤细胞后，溶瘤病毒大量扩增，并表达VEGFR ER-TPD元件，直接抑制肿瘤细胞VEGFR的表达，导致肿瘤细胞因缺乏足够的营养而生长抑制和凋亡。In some embodiments, 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.
在一些实施方案中，所述嵌合蛋白构建体的靶向结构域可以结合TGF-β1和PD-L1。任何能够靶向结合bcl-2的靶向结构域都可以使用，例如但不限于，利用TGF-β1和PD-L1抗体的重链和轻链重组的scFv串联ER-TPD元件，构建靶向TGF-β1和PD-L1ER-TPD元件，并克隆装载在溶瘤病毒强启动子后，特异性的感染肿瘤细胞后，溶瘤病毒大量扩增，并表达TGF-β1和PD-L1ER-TPD元件，直接抑制和降解TGF-β1和PD-L1在肿瘤细胞内的表达，打破肿瘤免疫抑制微环境，从而达到治疗肿瘤的效果。In some embodiments, the targeting domain of the chimeric protein construct can bind TGF-β1 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-β1 and PD-L1 antibodies to construct TGF-targeting -β1 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-β1 and PD-L1ER-TPD elements. Directly inhibit and degrade the expression of TGF-β1 and PD-L1 in tumor cells, break the tumor immunosuppressive microenvironment, and achieve the effect of treating tumors.
不受理论限制，但认为本申请的嵌合蛋白构建体通过溶瘤病毒载体在肿瘤细胞中表达具有独特的优势。一方面，通过本申请的嵌合蛋白构建体靶向降解肿瘤细胞中帮助肿瘤细胞生长或免疫逃逸或者对抗凋亡的蛋白，可以促使肿瘤细胞的凋亡或死亡；另一方面，溶瘤病毒载体可以在肿瘤细胞内特异性复制，通过病毒复制和裂解促使肿瘤细胞溶解，将两种不同的肿瘤杀伤手段整合在溶瘤病毒载体内，能够在同一个药物形式实现协同杀伤作用。Without being limited by theory, it is believed that the chimeric protein construct of the present application has unique advantages when expressed in tumor cells through oncolytic viral vectors. On the one hand, 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; on the other hand, 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.
在另一个方面，本公开还提供了一种药物组合物，其包含本公开所提供的溶瘤病毒和药学上可接受的媒介。在某些实施方式中，药学上可接受的媒介包含任何和所有运输媒介、溶剂、稀释剂、赋型剂、佐剂、分散介质、包被、抗菌剂和抗真菌剂、吸收剂等在哺乳动物和特别是人类对象中与施用兼容的此类物质。In another aspect, the present disclosure also provides a pharmaceutical composition comprising the oncolytic virus provided by the present disclosure and a pharmaceutically acceptable vehicle. In certain embodiments, 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.
在某些实施方式中，本申请所述的溶瘤病毒或其药物组合物配制为用于静脉内或瘤内施用。在某些实施方式中，溶瘤病毒或其药物组合物可被放置于适合人类或动物使用的溶剂或稀释剂中。所述溶剂或稀释剂优选为等渗的、低渗的或弱低渗的并且具有相对低的离子强度。典型示例包括无菌水、生理盐水(例如氯化钠)、林格液(Ringer’s solution)、葡萄糖、海藻糖或鹿糖溶液、Hank’s液和其他水性生理平衡盐溶液。In certain embodiments, the oncolytic viruses described herein, or pharmaceutical compositions thereof, are formulated for intravenous or intratumoral administration. In certain embodiments, 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.
在一个实施方式中，本申请所述的溶瘤病毒或其药物组合物可具有缓冲剂。合适的缓冲剂包括但不限于磷酸盐缓冲液(例如PBS)、碳酸氢盐缓冲液和/或能够保持生理上或微偏碱性的pH(例如从大约pH7到大约pH9)的Tris缓冲液。In one embodiment, 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).
在一个实施方式中，本发明的溶瘤病毒组合物可能为提高其稳定性特别是在生产条件下和长期(即至少6个月，优选至少2年)冰冻(例如-70℃、_20℃)储存、冷藏储存(例如4℃)、室温环境储存的稳定性而制备。多种本领 域既有的病毒制剂或为冰冻的液体形式或为冻干的形式(例如WO98/02522和WO2008/114021等)。固体(例如干粉或冻干)组合物能通过涉及真空干燥和冻干的步骤获得。为说明目的，加入NaCl和糖的缓冲制剂特别适于保存病毒。In one embodiment, 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. Various skills Existing viral preparations are either in frozen liquid form or in lyophilized form (eg WO98/02522 and WO2008/114021, etc.). Solid (eg dry powder or lyophilized) compositions can be obtained by steps involving vacuum drying and lyophilization. For illustrative purposes, buffer preparations with the addition of NaCl and sugar are particularly suitable for preserving viruses.
iii)细胞药物组合物iii) Cellular pharmaceutical compositions
在另一个方面，本公开还提供了一种细胞药物组合物，其包含本公开所提供的工程化细胞或细胞群和药学上可接受的媒介。示例性的媒介包括缓冲剂，如中性缓冲盐水、磷酸盐缓冲盐水等；碳水化合物，如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露糖醇；蛋白质；多肽或氨基酸，如甘氨酸；抗氧化剂；螯合剂，如EDTA或谷胱甘肽；佐剂(例如氢氧化铝)；和防腐剂。在一个方面，本发明的细胞药物组合物被配制用于静脉内施用。In another aspect, 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. In one aspect, the cellular pharmaceutical compositions of the invention are formulated for intravenous administration.
在一方面，本公开还提供了一种本申请所述的嵌合蛋白构建体、核酸分子或载体在制备待移植细胞(例如同种异体细胞移植)中的用途。In one aspect, the present disclosure also provides a use of the chimeric protein construct, nucleic acid molecule or vector described herein in preparing cells to be transplanted (eg, allogeneic cell transplantation).
在某些实施方式中，所述待移植细胞是免疫细胞(例如，T细胞)、干细胞(及其分化得到的细胞)、肾细胞、胰岛细胞、心肌细胞等。在某些实施方式中，所述待移植细胞可以是自体细胞，也可以是同种异体细胞。In certain embodiments, the cells to be transplanted are immune cells (eg, T cells), stem cells (and differentiated cells thereof), kidney cells, islet cells, cardiomyocytes, etc. In certain embodiments, the cells to be transplanted may be autologous cells or allogeneic cells.
在一方面，本申请还提供了一种表达上文所述的嵌合蛋白构建体、核酸分子或载体的待移植细胞，其中表达本申请所述的嵌合蛋白构建体，所述嵌合蛋白构建体包含ERAD机制蛋白结合结构域和靶向结构域，所述靶向结构域包含特异性靶向或结合靶蛋白的结构域，所述靶蛋白是移植排斥反应相关蛋白。在某些实施方式中，所述嵌合蛋白构建体进一步包含如本申请所述的蛋白降解通路成员(例如E3泛素连接酶、蛋白酶体、溶酶体)结合结构域。本申请所述的任何蛋白降解通路成员结合结构域都可以使用。In one aspect, 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. In certain embodiments, 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.
在某些实施方式中，所述移植排斥反应相关蛋白包括抗原呈递分子(例如MHC I类分子、MHC II类分子、MICA/B分子等)、抗原识别分子(例如TCR、CD123、NKG2D等)、免疫检查点分子(例如PD-1、PD-L1、CTLA4、TIM3,TIGIT,LAG3,A2AR,BTLA,IDO1,IDO2,TDO,KIR,NOX2,VISTA,SIGLEC7,PVR等)等。In certain embodiments, 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.
在某些实施方式中，所述移植排斥反应相关蛋白包含免疫原性蛋白(例如，HLA(HLAα/β)、TCR(αβTCR)、NKG2D(自然杀伤细胞2族成员D)配体等)。In certain embodiments, the transplant rejection-associated protein includes an immunogenic protein (eg, HLA (HLAα/β), TCR (αβTCR), NKG2D (natural killer cell family 2 member D) ligand, etc.).
本公开还提供上述待移植细胞及其治疗方法，能够克服现有细胞移植和细胞治疗中面临的个性化移植细胞和治疗细胞的来源问题，无法标准化和规模化生产，治疗效率低等问题。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.
免疫系统具有强大的可塑性和具有近乎无限的检测入侵病毒、细菌、异源细胞和病变细胞的能力。这种非凡的免疫监测能力主要通过体液免疫和细胞免疫实现的，其中包括两种重要分子结构：免疫球蛋白和T细胞受体(TCR)。TCR是T细胞的定义结构，是一种跨膜异源二聚体，由通过二硫键连接的α和β链或δ和γ链组成。在这些链中，互补决定区(CDR)决定了TCR将结合的抗原。在TCR中，TCRα和TCRβ亚基(或γδT细胞中的TCRγ和TCRδ)负责识别主要组织相容性复合体(MHC)/抗原配体。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). The TCR is the defining structure of T cells and is a transmembrane heterodimer composed of α and β chains or δ and γ chains linked by disulfide bonds. Within these chains, the complementarity determining regions (CDRs) determine the antigen to which the TCR will bind. Among TCRs, the TCRα and TCRβ subunits (or TCRγ and TCRδ in γδ T cells) are responsible for recognizing major histocompatibility complex (MHC)/antigen ligands.
人类MHCⅠ类基因区包括HLA-A、B、C位点的等位基因，编码HLA-A抗原、B抗原和C抗原等经典的Ⅰ类抗原(分子)，称为HLA-A、HLA-B和HLA-C。这些抗原分子存在于所有体细胞的表面，并与细胞内蛋白质表位肽段结合，供免疫系统进行识别。如果细胞产生突变蛋白，或有外来细菌或病毒入侵，细胞将这些突变蛋白或异源蛋白抗原表位呈递后，免疫细胞进行识别后会进行免疫攻击和杀伤，从而清除病变细胞、病菌和病毒入侵细胞。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. 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.
α/βT淋巴细胞通过称为αβT细胞抗原受体(TCR)CD3复合物的多聚蛋白集合识别肽-MHC配体。该结构由结合抗原的可变αβTCR二聚体和参与TCR.CD3表面转运、稳定和信号转导的三个不变二聚体(CD3γε、δε和ζζ)组成。αβT细胞受体(αβTCR)在大多数(约95％)T细胞上表达，并通过识别主要组织相容性复合体(MHC)锚定抗原在T细胞活化中发挥关键作用。因此，TCR介导的T细胞活化是同种异体造血细胞移植(allo-HCT)和同种异体CAR-T细胞治疗过程中移植物抗宿主病(GVHD)发病机制的关键步骤。α/β T lymphocytes recognize peptide-MHC ligands through a collection of polyproteins called the αβ T cell antigen receptor (TCR) CD3 complex. The structure consists of a variable αβTCR dimer that binds the antigen and three invariant dimers (CD3γε, δε, and ζζ) involved in TCR.CD3 surface transport, stabilization, and signal transduction. The αβ T cell receptor (αβTCR) is expressed on the majority (approximately 95%) of T cells and plays a key role in T cell activation by recognizing major histocompatibility complex (MHC) anchored antigens. Therefore, TCR-mediated T cell activation is a key step in the pathogenesis of graft-versus-host disease (GVHD) during allogeneic hematopoietic cell transplantation (allo-HCT) and allogeneic CAR-T cell therapy.
人类白细胞抗原(HLA)系统或复合物是一组由人类主要组织相容性复合物(MHC)基因复合物编码的相关蛋白质。这些细胞表面蛋白负责调节免疫系统。在治疗性移植环境中，当同种异体移植物上的供体HLA与受体不同时，就会发生“HLA不匹配”。HLA不匹配导致同种异体反应性T细胞的激活，这会在移植后的六个月内引起急性细胞排斥(ACR)。错配的供体HLA抗原也是新生供体特异性HLA抗体(dnDSA)开发的靶标，其在急性和慢性移植细胞(如T细胞)排斥中发挥增强作用。因此，为了产生用于安全同种异体输注和治疗目的的通用移植细胞(如T细胞)，建议通过基因破坏TCR来有效阻断移植物抗宿主病(GVHD)。此外，有必要抑制同种异体细胞(如T细胞)上的HLA表达，以减少受体免疫系统对同种异体T细胞TCRαβ和/或HLA I类的排斥反应。The human leukocyte antigen (HLA) system or complex is a group of related proteins encoded by the human major histocompatibility complex (MHC) gene complex. These cell surface proteins are responsible for regulating the immune system. In the therapeutic transplant setting, an "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. Therefore, to generate universal transplant cells (such as T cells) for safe allogeneic infusion and therapeutic purposes, it is recommended to effectively block graft-versus-host disease (GVHD) by genetically disrupting the TCR. In addition, it is necessary to suppress HLA expression on allogeneic cells, such as T cells, to reduce rejection by the recipient immune system of allogeneic T cells TCRαβ and/or HLA class I.
在某些实施方式中，表达上文所述的嵌合蛋白构建体、核酸分子或载体的待移植细胞具有降低的免疫原性。In certain embodiments, cells to be transplanted expressing the chimeric protein constructs, nucleic acid molecules or vectors described above have reduced immunogenicity.
在一些实施方案中，本申请所述的具有降低的免疫原性(或具有高度兼容性)的待移植细胞为T细胞，其表达的TPD嵌合蛋白构建体包含特异性识别TCR和/或HLA的scFv以及HCMV糖蛋白US2和/或US11的的跨膜结构域或其功能变体和内质网驻留结构域或其功能变体。In some embodiments, 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.
所述ERAD机制蛋白结合结构域包含如SEQ ID NO.112或SEQ ID NO.118所示的氨基酸序列或与其具有至少80％(例如，至少85％、至少90％、至少95％或至少99％)同一性的氨基酸序列。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.
在一些实施方案中，所述具有降低的免疫原性的待移植细胞为T细胞，其表达的ER-TPD嵌合蛋白构建体包含特异性识别TCR的scFv以及腺病毒E3-K19的跨膜结构域或其功能变体和内质网驻留结构域或其功能变体。在一些实施方案中，所述具有降低的免疫原性的待移植细胞为T细胞，其表达包含靶向蛋白结合结构域和ERAD机制蛋白结合结构域的ER-TPD嵌合蛋白构建体，所述靶向蛋白结合结构域包括包含于 如SEQ ID NO：152所示的氨基酸序列或与其具有至少80％(例如，至少85％、至少90％、至少95％或至少99％)同一性的氨基酸序列的6个CDR。所述ERAD机制蛋白结合结构域包含如SEQ ID NO.96所示的氨基酸序列或与其具有至少80％(例如，至少85％、至少90％、至少95％或至少99％)同一性的氨基酸序列。In some embodiments, 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. In some embodiments, 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. .
在一些实施方案中，上述具有降低的免疫原性的T细胞表达的ER-TPD嵌合蛋白构建体还包含蛋白降解通路成员(例如E3泛素连接酶、蛋白酶体)结合结构域，可选地，所述蛋白降解通路成员结合结构域与所述ERAD机制蛋白结合结构域连接。在一些实施方案中，上述具有降低的免疫原性的T细胞表达的ER-TPD嵌合蛋白构建体还包含具有如SEQ ID NO:162所示的氨基酸序列的E3泛素连接酶结合结构域。在一些实施方案中，上述具有降低的免疫原性的T细胞表达的ER-TPD嵌合蛋白构建体还包含具有如SEQ ID NO:166所示的氨基酸序列的E3泛素连接酶结合结构域。In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.
在一些实施方案中，上述具有降低的免疫原性的待移植细胞(如T细胞)还表达如上文所述的共表达部分。In some embodiments, the cells to be transplanted (eg, T cells) with reduced immunogenicity also express a co-expression moiety as described above.
例如，为了降低细胞的免疫原性或抗原呈递，可以在上述具有降低的免疫原性的待移植细胞(如T细胞)中表达能够降解或减少MHC I类或II类分子的蛋白作为共表达部分(例如，完整的病毒ER驻留糖蛋白，包括但不限于，HCMV US2、US3、US11、US10、腺病毒E3-K19、HCMV US6和HSV ICP47)。TCR的有效下调将大大抑制TCR介导的免疫攻击，减少异体输注T细胞时的GVHD。包括天然病毒ER驻留的糖蛋白可以进一步抑制MHC分子，从而阻止肽呈递给受体CD8+T细胞，抑制异体T细胞的免疫识别。因此，如上文所述的高度兼容的T细胞也称为“自体通用T细胞(Universal T cell,UT)”或“通用T细胞(Universal T cell,UT)”，可实现异体输血治疗目的，且可以提高异体T细胞的兼容性和输注后的长期持久性。For example, in order to reduce the immunogenicity or antigen presentation of cells, 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. (For example, 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.
在另一方面，本公开提供了一种降低受试者对细胞移植免疫反应的方法，包括向受试者施用治疗有效量的如上文所述的具有降低的免疫原性的待移植细胞，从而降低受试者对所述移植物或对所述经修饰的细胞的免疫反应。在某些实施方式中，所述待移植细胞在细胞内表达如本文所述的TPD嵌合蛋白构建体，所述TPD嵌合蛋白构建体包含靶向免疫原性蛋白(例如，HLA(HLAα/β)、TCR(αβTCR)、NKG2D(自然杀伤细胞2族成员D)配体等)的结构域以及上文所述的ERAD机制蛋白结合结构域，以及可选地，蛋白降解通路成员结合结构域。In another aspect, 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. In certain embodiments, 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 (HLAα/ β), TCR (αβTCR), 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 .
1.细胞治疗1. Cell therapy
细胞治疗，通过基因工程技术改造细胞(例如免疫细胞)使其能呈递肿瘤相关抗原或表达特异性识别疾病细胞的受体，在体外指数级扩增后回输至患者体内，激活体内免疫系统对肿瘤细胞进行攻击或直接特异性识别并杀伤疾病细胞的疗效。2012年CD19靶向CAR-T细胞实现了人类医学史上首次定向清除B细胞白血病患者体内肿瘤细胞，成为继骨髓干细胞移植治疗技术后又一项能真正治愈白血病的治疗技术，开启了精准医学细胞治疗的新时代。这项技术有望应用于各种血液肿瘤和实体瘤的治疗上。但目前常规CAR-T治疗实体肿瘤的临床疗效不佳，其原因：(1)CAR-T的杀伤功能高度依赖于CAR结构对肿瘤相关抗原(TAA)的识别，但由于实体肿瘤的异质性，肿瘤细胞表面靶点蛋白表达存在很大差异；目前单一靶向的CAR-T无法完全识别和杀伤所有肿瘤细胞，从而导致肿瘤的免疫逃逸和复发转移；(2)实体肿瘤的直接免疫抑制效应，表达PD-L1，B3H7等，直接抑制T细胞的激活；(3)实体肿瘤的免疫抑制微环境，有大量的免疫抑制细胞，如TAM、Treg、MDSC，传统CAR-T的设计很难突破肿瘤内部的免疫抑制细胞的限制；(4)传统CAR-T是个性化的细胞治疗，T细胞来源患者本身，如果患者已经接受大量的放化疗等，免疫系统功能受损，很难从患者外周血分离出足够数量的T细胞，即使分离出来改造，T细胞的增殖和杀伤功能还是很薄弱，因此很难发挥治疗效果。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. In 2012, 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. However, the current clinical efficacy of conventional CAR-T in the treatment of solid tumors is poor. The reasons are: (1) The killing function of CAR-T is highly dependent on the recognition of tumor-associated antigens (TAA) by the CAR structure. However, due to the heterogeneity of solid tumors, , there are great differences in the expression of target proteins on the surface of tumor cells; the current single-targeted CAR-T cannot completely recognize and kill all tumor cells, leading to immune escape and recurrence and metastasis of the tumor; (2) direct immunosuppressive effect of solid tumors , expressing PD-L1, B3H7, etc., directly inhibiting the activation of T cells; (3) The immunosuppressive microenvironment of solid tumors has a large number of immunosuppressive cells, such as TAM, Treg, and MDSC, and it is difficult to break through the traditional CAR-T design. Limitation of immunosuppressive cells within the tumor; (4) Traditional CAR-T is a personalized cell therapy, and the 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.
在本申请中提供的待移植的细胞(例如T细胞)，能够克服现有细胞治疗中面临的肿瘤靶标差异性，肿瘤免疫抑制环境，个性化治疗细胞来源等问题。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.
为了细胞治疗的目的，可以进一步对具有降低的免疫原性的待移植细胞(例如T细胞，进一步地，具有TCRαβ和MHC分子表面表达缺陷的通用T细胞)进行基因工程改造，例如以表达如上文所述的共表达部分，以达到治疗目的。For the purpose of cell therapy, the cells to be transplanted with reduced immunogenicity (such as T cells, further, universal T cells with defective surface expression of TCRαβ and MHC molecules) can be further genetically engineered, for example to express as above The co-expression part is used to achieve therapeutic purposes.
例如，为了使待移植细胞(如T细胞)能够特异性作用于疾病相关的靶蛋白或表达疾病相关的靶蛋白的细胞，可以在上述具有降低的免疫原性的待移植细胞(如T细胞)中表达识别或结合疾病相关的靶蛋白的结合结构域或分子，例如嵌合抗原受体CAR，工程化TCR、或本申请提供的CNK受体、或抗病毒蛋白结合结构域等。For example, in order to enable the cells to be transplanted (such as T cells) to specifically act on disease-related target proteins or cells expressing disease-related target proteins, the cells to be transplanted (such as T cells) with reduced immunogenicity 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.
在一些实施方式中，也可以在上述具有降低的免疫原性的待移植细胞(如T细胞)中表达能够介导待移植疾病迁移至疾病微环境的分子(例如趋化因子受体或细胞因子受体)。In some embodiments, molecules capable of mediating the migration of the disease to be transplanted to the disease microenvironment (such as chemokine receptors or cytokines) can also be expressed in the cells to be transplanted (such as T cells) with reduced immunogenicity. receptor).
再例如，为了使待移植细胞(如T细胞)能够具有增加的免疫刺激活性，可以在上述具有降低的免疫原性的待移植细胞(如T细胞)中表达免疫刺激性分子。For another example, in order to enable the cells to be transplanted (such as T cells) to have increased immunostimulatory activity, immunostimulatory molecules can be expressed in the cells to be transplanted (such as T cells) with reduced immunogenicity.
在另一方面，本申请还提供了在本申请提供的待移植细胞(例如UT细胞)中通过导入NK元件，特别是优化重组的NK元件，让T细胞能象NK细胞一样高效广谱的识别所有病毒感染细胞和肿瘤细胞，可选地，进一步通过优化转导元件，使得CNK-T细胞能高效激活并杀伤肿瘤细胞。因为NK靶点包括MICA、MICB和ULBP1–6等家族成员蛋白，能广泛表达在各类肿瘤细胞并覆盖肿瘤进展的不同阶段，所以，CNK技术可有效的解决单一CAR-T的脱靶效应，消除肿瘤免疫逃逸的机率。同时，CNK在设计中导入的复合型转接器(Chimeric Adaptor)，能有效的转导和放大NK信号，跨越如PD1信号的免疫检查点的限制，高效激活T细胞，并实现对肿瘤细胞的杀伤。复合型转接器(Chimeric Adaptor)实现放大CNK-T细胞信号，能抵抗肿瘤环境中的免疫抑制，实现对T细胞 的激活，实现对肿瘤细胞的杀伤；再有CNK-T细胞通过NK识别靶点，还能清除MDSC等免疫抑制细胞。病毒感染细胞后，表达特定功能蛋白，MHCI的组装或转运或直接促进MHCI分子的定向降解，从而抑制病毒抗原表位的呈递，产生免疫逃逸。On the other hand, 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. For all virus-infected cells and tumor cells, optionally, transduction elements are further optimized so that CNK-T cells can efficiently activate and kill tumor cells. Because NK targets include family member proteins such as MICA, MICB, and ULBP1–6, which can be widely expressed in various types of tumor cells and cover different stages of tumor progression, CNK technology can effectively solve the off-target effects of a single CAR-T and eliminate The probability of tumor immune escape. At the same time, 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. 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.
在一方面，本公开提供了一种UT细胞，其还表达CNK受体以及可选地CAR，这种UT细胞也可称为“CNKT-UT”。在本申请中验证了CNK-UT具有广谱的肿瘤识别和杀伤能力。本申请进一步设计复合特定靶点CAR/CNK-UT产品，论证了CAR/CNK-UT产品比常规CAR-T对肿瘤细胞具有更强大的杀伤和激活功能；在动物实验，CAR/CNK-UT产品也具有更高效的肿瘤清除能力。UT技术实现了异体通用型的改造，T细胞来源健康供者，从而实现了CNK-UT产品的标准化和规模化生产，可以提前制备，并保证T细胞对肿瘤细胞杀伤和激活的功能。In one aspect, 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." In this application, it was verified that 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.
在一些优选的实施方案中，所述CNKT-UT包含一种核酸分子，其编码与如SEQ ID NO.158所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列。在一些优选的实施方案中，其中所述CNKT-UT包含一种核酸分子，其编码如SEQ ID NO.158所示的氨基酸序列。在另一方面，本公开还提供了CNKT-UT在制备用于治疗疾病的药物中的用途。In some preferred embodiments, the CNKT-UT comprises a nucleic acid molecule encoding an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 158, preferably 85%, 90 %, 95%, 96%, 97%, 98%, 99% or above identical amino acid sequences. In some preferred embodiments, wherein the CNKT-UT comprises a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 158. In another aspect, the present disclosure also provides the use of CNKT-UT in the preparation of a medicament for treating a disease.
在一方面，本公开还提供了一种包含或表达本申请所述的嵌合蛋白构建体、编码其核酸分子或载体的免疫细胞，其中所述嵌合蛋白构建体包含ERAD机制蛋白结合结构域和靶向结构域，所述靶向结构域包含特异性靶向或结合靶蛋白的结构域，所述靶蛋白在所述免疫细胞上以及在靶细胞上都有表达。在某些实施方式中，所述免疫细胞进一步包含或表达靶向所述靶蛋白的结合结构域或分子(例如CAR)、编码其的核酸分子或载体。在某些实施方式中，此类靶蛋白的例子包括，CD123、CD5、CD7、CD38、或CD4。In one aspect, 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. In certain embodiments, 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. In certain embodiments, examples of such target proteins include CD123, CD5, CD7, CD38, or CD4.
不受理论限制，某些靶蛋白既在靶细胞(例如癌细胞或病毒感染细胞)上表达，又在用于治疗的免疫细胞上表达。这类靶点通常难以通过细胞治疗的方法进行治疗，因此当用于治疗的免疫细胞上表达靶向此类靶点的CAR，则CAR也会导致识别免疫细胞自身表达的同一靶点，导致免疫细胞发生自噬，进而无法正常扩增，或者对靶细胞杀伤效果有限。通过本申请提供的嵌合蛋白构建体，可以将免疫细胞自身表达的靶蛋白降解，从而避免自噬作用，同时又保留了对靶细胞的特异性杀伤作用。使用本申请的嵌合蛋白构建体的另一个优势在于，可以通过共表达的方式同时表达靶向该靶点的嵌合蛋白构建体和靶向该靶点的结合结构域(例如CAR、TCR等)，比传统基因敲除等方法更简便，效果更好。Without being bound by theory, certain 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. Through the chimeric protein construct provided in this application, 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.
在某些实施方式中，所述嵌合蛋白构建体进一步包含如本申请所述的蛋白降解通路成员(例如E3泛素连接酶、蛋白酶体、溶酶体)结合结构域。本申请所述的任何蛋白降解通路成员结合结构域都可以使用。In certain embodiments, 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.
用于过继细胞疗法的细胞施用方法是已知的，并且可以与所提供的方法和组合物结合使用。例如，过继性T细胞疗法的方法在例如Gruenberg等的美国专利申请公开号2003/0170238；Rosenberg的美国专利号4,690,915；Rosenberg(2011)Nat Rev Clin Oncol.8(10):577-85)中描述。参见例如Themeli等(2013)Nat Biotechnol.31(10):928-933；Tsukahara等(2013)Biochem Biophys Res Commun 438(1):84-9；Davila等(2013)PLoS ONE 8(4):e61338。Cell administration methods for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, methods of adoptive T cell therapy are 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) . See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10):928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9; Davila et al. (2013) PLoS ONE 8(4):e61338 .
在某些实施方式中，将细胞或细胞的个体群体或亚型以约一百万至约一千亿个细胞的范围施用于受试者，例如100万至约500亿个细胞(例如约500万个细胞、约2500万个细胞、约5亿个细胞、约10亿个细胞、约50亿个细胞、约200亿个细胞、约300亿个细胞、约400亿个细胞、或任意上述两个值定义的范围)，例如约1000万到约1000亿个细胞(例如约2000万个细胞、约3000万个细胞、约4000万个细胞、约6000万个细胞、约7000万个细胞、约8000万个细胞、约9000万个细胞、约100亿个细胞、约250亿个细胞、约500亿个细胞、约750亿个细胞、约900亿个细胞，或任意上述两个值定义的范围)，和在某些情况下，约1亿个细胞至约500亿个细胞(例如，约1.2亿个细胞、约2.5亿个细胞、约3.5亿个细胞、约4.5亿个细胞、约6.5亿个细胞、约8亿个细胞、约9亿个细胞、约30亿个细胞、约300亿个细胞、约450亿个细胞)或这些范围之间的任何值。In certain embodiments, 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, about 30 billion cells, about 45 billion cells) or any value between these ranges.
在一些实施方式中，总细胞的剂量和/或细胞的个体亚群的剂量在处于或大约104个细胞/千克(kg)体重至处于或大约109个细胞/千克(kg)体重之间的范围内，例如在105和106个细胞/kg体重之间，例如，至少或至少大约或处于或大约1×105个细胞/kg、1.5×105个细胞/kg，2×105个细胞/kg或1×106个细胞/kg体重。例如，在一些实施方式中，细胞以在处于或大约104和处于或约109个T细胞/千克(kg)体重之间或其一定误差范围内施用，例如在105和106个T细胞/kg体重之间，例如至少或至少大约或处于或大约1×105个T细胞/kg、1.5×105个T细胞/kg，2×105个T细胞/kg、或1×106个T细胞/kg体重。In some embodiments, 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. For example, in some embodiments, 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.
细胞可以通过任何合适的方式来施用，例如通过推注，通过注射，例如静脉内或皮下注射、眼内注射、眼周注射、视网膜下注射、玻璃体内注射、经中隔注射、巩膜下注射、脉络膜内注射、前房内注射、会阴下注射、结膜下注射、眼球筋膜囊下(sub-Tenon)注射、球后注射、球周注射，或后近巩膜(posterior juxtascleral)递送。在一些实施方式中，它们通过肠胃外、肺内和鼻内施用，并且如果局部治疗是期望的，则通过病灶内施用。肠胃外输注包括肌内、静脉内、动脉内、腹膜内或皮下给药。在一些实施方式中，给定剂量通过单次推注施用细胞来施用。在一些实施方式中，其通过多次推注施用细胞来施用，例如，在不超过3天的期间，或通过连续输注施用细胞来施用。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. In some embodiments, 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. In some embodiments, 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.
在一些实施方式中，提供了重复剂量方法，其中给予第一剂量的细胞，然后给予一个或多个第二连续剂量。当以过继治疗方法施用于受试者时，通常设计细胞的多剂量的时机和大小以增加表达抗原的T细胞(例如表达CAR的T细胞)的功效和/或活性和/或功能。在一些实施方式中，重复给药减少了当抑制性免疫分子例如PD-1和/ 或PD-L1在表达抗原的例如表达CAR的T细胞上被上调时可发生的下调或抑制活性。方法包括施用第一剂量，通常随后是一个或多个连续剂量，并且在不同剂量之间具有特定的时间范围。In some embodiments, repeated dosing methods are provided wherein a first dose of cells is administered followed by one or more second consecutive doses. When administered to a subject in an adoptive therapy approach, 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). In some embodiments, 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.
在过继细胞疗法的背景下，给定“剂量”的施用包括作为单一组合物和/或单次不间断施用(例如，作为单次注射或连续输注)的给定量或数量的细胞的施用，并且还包括在指定的时间段(不超过3天)中，以多个个体组合物或输注提供的给定量或数量的细胞数量作为分次剂量的施用。因此，在某些情况下，第一或连续剂量是在单个时间点给予或启动的指定数量细胞的单次或连续施用。然而，在某些情况下，第一或连续剂量在不超过三天的时间段内以多次注射或输注施用，例如三天或两天、每天一次，或在一天时间内多次输注。In the context of adoptive cell therapy, administration of a given "dose" includes administration of a given amount or number of cells as a single composition and/or 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). Thus, in some cases, 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. However, in some cases, 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. .
细胞治疗，通过基因工程技术改造细胞(例如免疫细胞)使其能呈递肿瘤相关抗原或表达特异性识别疾病细胞的受体，在体外指数级扩增后回输至患者体内，激活体内免疫系统对肿瘤细胞进行攻击或直接特异性识别并杀伤疾病细胞的疗效。2012年CD19靶向CAR-T细胞实现了人类医学史上首次定向清除B细胞白血病患者体内肿瘤细胞，成为继骨髓干细胞移植治疗技术后又一项能真正治愈白血病的治疗技术，开启了精准医学细胞治疗的新时代。这项技术有望应用于各种血液肿瘤和实体瘤的治疗上。但目前常规CAR-T治疗实体肿瘤的临床疗效不佳，其原因：(1)CAR-T的杀伤功能高度依赖于CAR结构对肿瘤相关抗原(TAA)的识别，但由于实体肿瘤的异质性，肿瘤细胞表面靶点蛋白表达存在很大差异；目前单一靶向的CAR-T无法完全识别和杀伤所有肿瘤细胞，从而导致肿瘤的免疫逃逸和复发转移；(2)实体肿瘤的直接免疫抑制效应，表达PD-L1，B3H7等，直接抑制T细胞的激活；(3)实体肿瘤的免疫抑制微环境，有大量的免疫抑制细胞，如TAM、Treg、MDSC，传统CAR-T的设计很难突破肿瘤内部的免疫抑制细胞的限制；(4)传统CAR-T是个性化的细胞治疗，T细胞来源患者本身，如果患者已经接受大量的放化疗等，免疫系统功能受损，很难从患者外周血分离出足够数量的T细胞，即使分离出来改造，T细胞的增殖和杀伤功能还是很薄弱，因此很难发挥治疗效果。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. In 2012, 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. However, the current clinical efficacy of conventional CAR-T in the treatment of solid tumors is poor. The reasons are: (1) The killing function of CAR-T is highly dependent on the recognition of tumor-associated antigens (TAA) by the CAR structure. However, due to the heterogeneity of solid tumors, , there are great differences in the expression of target proteins on the surface of tumor cells; the current single-targeted CAR-T cannot completely recognize and kill all tumor cells, leading to immune escape and recurrence and metastasis of the tumor; (2) direct immunosuppressive effect of solid tumors , expressing PD-L1, B3H7, etc., directly inhibiting the activation of T cells; (3) The immunosuppressive microenvironment of solid tumors has a large number of immunosuppressive cells, such as TAM, Treg, and MDSC, and it is difficult to break through the traditional CAR-T design. Limitation of immunosuppressive cells within the tumor; (4) Traditional CAR-T is a personalized cell therapy, and the 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.
在本申请中提供的待移植的细胞(例如T细胞)，能够克服现有细胞治疗中面临的肿瘤靶标差异性，肿瘤免疫抑制环境，个性化治疗细胞来源等问题。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.
为了细胞治疗的目的，可以进一步对具有降低的免疫原性的待移植细胞(例如T细胞，进一步地，具有TCRαβ和MHC分子表面表达缺陷的通用T细胞)进行基因工程改造，例如以表达如上文所述的共表达部分，以达到治疗目的。For the purpose of cell therapy, the cells to be transplanted with reduced immunogenicity (such as T cells, further, universal T cells with defective surface expression of TCRαβ and MHC molecules) can be further genetically engineered, for example to express as above The co-expression part is used to achieve therapeutic purposes.
例如，为了使待移植细胞(如T细胞)能够特异性作用于疾病相关的靶蛋白或表达疾病相关的靶蛋白的细胞，可以在上述具有降低的免疫原性的待移植细胞(如T细胞)中表达识别或结合疾病相关的靶蛋白的结合结构域或分子，例如嵌合抗原受体CAR，工程化TCR、或本申请提供的CNK受体、或抗病毒蛋白结合结构域等。For example, in order to enable the cells to be transplanted (such as T cells) to specifically act on disease-related target proteins or cells expressing disease-related target proteins, the cells to be transplanted (such as T cells) with reduced immunogenicity 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.
在一些实施方式中，也可以在上述具有降低的免疫原性的待移植细胞(如T细胞)中表达能够介导待移植疾病迁移至疾病微环境的分子(例如趋化因子受体或细胞因子受体)。In some embodiments, molecules capable of mediating the migration of the disease to be transplanted to the disease microenvironment (such as chemokine receptors or cytokines) can also be expressed in the cells to be transplanted (such as T cells) with reduced immunogenicity. receptor).
再例如，为了使待移植细胞(如T细胞)能够具有增加的免疫刺激活性，可以在上述具有降低的免疫原性的待移植细胞(如T细胞)中表达免疫刺激性分子。For another example, in order to enable the cells to be transplanted (such as T cells) to have increased immunostimulatory activity, immunostimulatory molecules can be expressed in the cells to be transplanted (such as T cells) with reduced immunogenicity.
在另一方面，本申请还提供了在本申请提供的待移植细胞(例如UT细胞)中通过导入NK元件，特别是优化重组的NK元件，让T细胞能象NK细胞一样高效广谱的识别所有病毒感染细胞和肿瘤细胞，可选地，进一步通过优化转导元件，使得CNK-T细胞能高效激活并杀伤肿瘤细胞。因为NK靶点包括MICA、MICB和ULBP1–6等家族成员蛋白，能广泛表达在各类肿瘤细胞并覆盖肿瘤进展的不同阶段，所以，CNK技术可有效的解决单一CAR-T的脱靶效应，消除肿瘤免疫逃逸的机率。同时，CNK在设计中导入的复合型转接器(Chimeric Adaptor)，能有效的转导和放大NK信号，跨越如PD1信号的免疫检查点的限制，高效激活T细胞，并实现对肿瘤细胞的杀伤。复合型转接器(Chimeric Adaptor)实现放大CNK-T细胞信号，能抵抗肿瘤环境中的免疫抑制，实现对T细胞的激活，实现对肿瘤细胞的杀伤；再有CNK-T细胞通过NK识别靶点，还能清除MDSC等免疫抑制细胞。病毒感染细胞后，表达特定功能蛋白，MHCI的组装或转运或直接促进MHCI分子的定向降解，从而抑制病毒抗原表位的呈递，产生免疫逃逸。On the other hand, 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. For all virus-infected cells and tumor cells, optionally, transduction elements are further optimized so that CNK-T cells can efficiently activate and kill tumor cells. Because NK targets include family member proteins such as MICA, MICB, and ULBP1–6, which can be widely expressed in various types of tumor cells and cover different stages of tumor progression, CNK technology can effectively solve the off-target effects of a single CAR-T and eliminate The probability of tumor immune escape. At the same time, 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.
在一方面，本公开提供了一种UT细胞，其还表达CNK受体以及可选地CAR，这种UT细胞也可称为“CNKT-UT”。在本申请中验证了CNK-UT具有广谱的肿瘤识别和杀伤能力。本申请进一步设计复合特定靶点CAR/CNK-UT产品，论证了CAR/CNK-UT产品比常规CAR-T对肿瘤细胞具有更强大的杀伤和激活功能；在动物实验，CAR/CNK-UT产品也具有更高效的肿瘤清除能力。UT技术实现了异体通用型的改造，T细胞来源健康供者，从而实现了CNK-UT产品的标准化和规模化生产，可以提前制备，并保证T细胞对肿瘤细胞杀伤和激活的功能。In one aspect, 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." In this application, it was verified that 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.
在一些优选的实施方案中，所述CNKT-UT包含一种核酸分子，其编码与如SEQ ID NO.158所示的氨基酸序列有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列。在一些优选的实施方案中，其中所述CNKT-UT包含一种核酸分子，其编码如SEQ ID NO.158所示的氨基酸序列。在另一方面，本公开还提供了CNKT-UT在制备用于治疗疾病的药物中的用途。In some preferred embodiments, the CNKT-UT comprises a nucleic acid molecule encoding an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 158, preferably 85%, 90 %, 95%, 96%, 97%, 98%, 99% or above identical amino acid sequences. In some preferred embodiments, wherein the CNKT-UT comprises a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 158. In another aspect, the present disclosure also provides the use of CNKT-UT in the preparation of a medicament for treating a disease.
在一方面，本公开还提供了一种包含或表达本申请所述的嵌合蛋白构建体、编码其核酸分子或载体的免疫细胞，其中所述嵌合蛋白构建体包含ERAD机制蛋白结合结构域和靶向结构域，所述靶向结构域包含特异性靶向或结合靶蛋白的结构域，所述靶蛋白在所述免疫细胞上以及在靶细胞上都有表达。在某些实施方式中，所述免疫细胞进一步包含或表达靶向所述靶蛋白的结合结构域或分子(例如CAR)、编码其的核酸分子或载体。在某些实施方 式中，此类靶蛋白的例子包括，CD123，CD5，CD7，CD38，或CD4。In one aspect, 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. In certain embodiments, 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. In some embodiments In the formula, examples of such target proteins include CD123, CD5, CD7, CD38, or CD4.
不受理论限制，某些靶蛋白既在靶细胞(例如癌细胞或病毒感染细胞)上表达，又在用于治疗的免疫细胞上表达。这类靶点通常难以通过细胞治疗的方法进行治疗，因此当用于治疗的免疫细胞上表达靶向此类靶点的CAR，则CAR也会导致识别免疫细胞自身表达的同一靶点，导致免疫细胞发生自噬，进而无法正常扩增，或者对靶细胞杀伤效果有限。通过本申请提供的嵌合蛋白构建体，可以将免疫细胞自身表达的靶蛋白降解，从而避免自噬作用，同时又保留了对靶细胞的特异性杀伤作用。使用本申请的嵌合蛋白构建体的另一个优势在于，可以通过共表达的方式同时表达靶向该靶点的嵌合蛋白构建体和靶向该靶点的结合结构域(例如CAR,TCR等)，比传统基因敲除等方法更简便，效果更好。Without being bound by theory, certain 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. Through the chimeric protein construct provided in this application, 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.
在某些实施方式中，所述嵌合蛋白构建体进一步包含如本申请所述的蛋白降解通路成员(例如E3泛素连接酶、蛋白酶体、溶酶体)结合结构域。本申请所述的任何蛋白降解通路成员结合结构域都可以使用。In certain embodiments, 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.
用于过继细胞疗法的细胞施用方法是已知的，并且可以与所提供的方法和组合物结合使用。例如，过继性T细胞疗法的方法在例如Gruenberg等的美国专利申请公开号2003/0170238；Rosenberg的美国专利号4,690,915；Rosenberg(2011)Nat Rev Clin Oncol.8(10):577-85)中描述。参见例如Themeli等(2013)Nat Biotechnol.31(10):928-933；Tsukahara等(2013)Biochem Biophys Res Commun 438(1):84-9；Davila等(2013)PLoS ONE 8(4):e61338。Cell administration methods for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, methods of adoptive T cell therapy are 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) . See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10):928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9; Davila et al. (2013) PLoS ONE 8(4):e61338 .
在某些实施方式中，将细胞或细胞的个体群体或亚型以约一百万至约一千亿个细胞的范围施用于受试者，例如100万至约500亿个细胞(例如约500万个细胞、约2500万个细胞、约5亿个细胞、约10亿个细胞、约50亿个细胞、约200亿个细胞、约300亿个细胞、约400亿个细胞、或任意上述两个值定义的范围)，例如约1000万到约1000亿个细胞(例如约2000万个细胞、约3000万个细胞、约4000万个细胞、约6000万个细胞、约7000万个细胞、约8000万个细胞、约9000万个细胞、约100亿个细胞、约250亿个细胞、约500亿个细胞、约750亿个细胞、约900亿个细胞，或任意上述两个值定义的范围)，和在某些情况下，约1亿个细胞至约500亿个细胞(例如，约1.2亿个细胞、约2.5亿个细胞、约3.5亿个细胞、约4.5亿个细胞、约6.5亿个细胞、约8亿个细胞、约9亿个细胞、约30亿个细胞、约300亿个细胞、约450亿个细胞)或这些范围之间的任何值。In certain embodiments, 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, about 30 billion cells, about 45 billion cells) or any value between these ranges.
在一些实施方式中，总细胞的剂量和/或细胞的个体亚群的剂量在处于或大约104个细胞/千克(kg)体重至处于或大约109个细胞/千克(kg)体重之间的范围内，例如在105和106个细胞/kg体重之间，例如，至少或至少大约或处于或大约1×105个细胞/kg、1.5×105个细胞/kg，2×105个细胞/kg或1×106个细胞/kg体重。例如，在一些实施方式中，细胞以在处于或大约104和处于或约109个T细胞/千克(kg)体重之间或其一定误差范围内施用，例如在105和106个T细胞/kg体重之间，例如至少或至少大约或处于或大约1×105个T细胞/kg、1.5×105个T细胞/kg，2×105个T细胞/kg、或1×106个T细胞/kg体重。In some embodiments, 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. For example, in some embodiments, 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.
细胞可以通过任何合适的方式来施用，例如通过推注，通过注射，例如静脉内或皮下注射、眼内注射、眼周注射、视网膜下注射、玻璃体内注射、经中隔注射、巩膜下注射、脉络膜内注射、前房内注射、会阴下注射、结膜下注射、眼球筋膜囊下(sub-Tenon)注射、球后注射、球周注射，或后近巩膜(posterior juxtascleral)递送。在一些实施方式中，它们通过肠胃外、肺内和鼻内施用，并且如果局部治疗是期望的，则通过病灶内施用。肠胃外输注包括肌内、静脉内、动脉内、腹膜内或皮下给药。在一些实施方式中，给定剂量通过单次推注施用细胞来施用。在一些实施方式中，其通过多次推注施用细胞来施用，例如，在不超过3天的期间，或通过连续输注施用细胞来施用。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. In some embodiments, 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. In some embodiments, 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.
在一些实施方式中，提供了重复剂量方法，其中给予第一剂量的细胞，然后给予一个或多个第二连续剂量。当以过继治疗方法施用于受试者时，通常设计细胞的多剂量的时机和大小以增加表达抗原的T细胞(例如表达CAR的T细胞)的功效和/或活性和/或功能。在一些实施方式中，重复给药减少了当抑制性免疫分子例如PD-1和/或PD-L1在表达抗原的例如表达CAR的T细胞上被上调时可发生的下调或抑制活性。方法包括施用第一剂量，通常随后是一个或多个连续剂量，并且在不同剂量之间具有特定的时间范围。In some embodiments, repeated dosing methods are provided wherein a first dose of cells is administered followed by one or more second consecutive doses. When administered to a subject in an adoptive therapy approach, 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). In some embodiments, 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.
在过继细胞疗法的背景下，给定“剂量”的施用包括作为单一组合物和/或单次不间断施用(例如，作为单次注射或连续输注)的给定量或数量的细胞的施用，并且还包括在指定的时间段(不超过3天)中，以多个个体组合物或输注提供的给定量或数量的细胞数量作为分次剂量的施用。因此，在某些情况下，第一或连续剂量是在单个时间点给予或启动的指定数量细胞的单次或连续施用。然而，在某些情况下，第一或连续剂量在不超过三天的时间段内以多次注射或输注施用，例如三天或两天、每天一次，或在一天时间内多次输注。In the context of adoptive cell therapy, administration of a given "dose" includes administration of a given amount or number of cells as a single composition and/or 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). Thus, in some cases, 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. However, in some cases, 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. .
在另一方面，本公开还提供了一种在体内或体外降解靶蛋白的方法，其包括将如本文所述的核酸分子和/或载体递送至表达靶蛋白的细胞或受试者内，所述核酸分子和/或所述载体在所述细胞或受试者内表达产生如本文所述的嵌合蛋白构建体。所述嵌合蛋白构建体的靶向蛋白结合结构域能够结合靶蛋白，所述嵌合蛋白构建体的ERAD机制蛋白结合结构域能够利用ERAD通路诱导泛素-蛋白酶体系统(UPS)的空间上靠近，从而利用UPS中的蛋白酶体来降解靶蛋白。当所述嵌合蛋白构建体进一步包含蛋白降解通路成员结合结构域时，该方法将诱导额外的降解系统靠近靶蛋白，使得降解效果得到显著提升。In another aspect, 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. When 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.
在另一方面，本公开还提供了一种治疗有需要的受试者的病症或疾病和/或预防其复发的方法，包括：向所述受试者施用治疗有效量的上文所述的药物组合物(包含例如本申请所述的核酸分子，例如mRNA、病毒载体、溶瘤病毒、嵌合蛋白构建体等)。In another aspect, 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.).
在某些实施方式中，所述方法包括将本申请所述的编码嵌合蛋白构建体的核酸分子(例如mRNA)或载体 (例如病毒载体)导入有需要的个体中，以使所述核酸分子或载体在所述个体的细胞中表达所述嵌合蛋白构建体。In certain embodiments, 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.
任何常规的施用路径都可在本发明的情况下使用，包括非肠道的、局部的或粘膜路径。非肠道途径为注射或输液施用，包含全身以及局部路径。通常非肠道注射类型是静脉内的(进入静脉)、动脉内的(进入动脉)、皮肤内的(进入真皮层)、皮下的(表皮以下)、肌肉的(进入肌肉)以及瘤内的(进入肿瘤或极为贴近肿瘤的部位)。典型的输液是通过静脉途径。粘膜施用包含但不限于口/食道、鼻内、气管、肺内、阴道内、或直肠内的途径。局部施用也可通过透皮肤的手段完成(例如贴片等依此类推)。可使用常规的注射器和针头来施用或任何现有技术中已有的能够促进或提高在对象内递送活性剂的化合物或装置。Any conventional route of administration may be used in the context of the present invention, including parenteral, topical or mucosal routes. 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). Typically 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.
根据所述个体需要治疗的疾病，以及需要降解的致病蛋白，可以选择适当的嵌合蛋白构建体，及其核酸分子和载体。Depending on the disease that the individual needs to treat and the disease-causing protein that needs to be degraded, the appropriate chimeric protein construct, its nucleic acid molecule and its vector can be selected.
在一些优选的实施方案中，所述疾病包括各类实体肿瘤和血液肿瘤、感染性疾病(例如病毒感染类疾病)、自身免疫性疾病、神经退行性疾病、代谢类疾病。In some preferred embodiments, 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.
在一些实施方案中，所述疾病是肿瘤或癌症，所述嵌合蛋白构建体的靶向结构域包含特异性靶向肿瘤相关的靶点或免疫功能相关的靶点。所述肿瘤相关的靶点或免疫功能相关的靶点如本申请前文所述。In some embodiments, the disease is a tumor or cancer, and 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.
在一些优选的实施方案中，所述实体肿瘤选自神经系统肿瘤、头颈部肿瘤、胸部肿瘤、消化系统肿瘤、泌尿生殖系统肿瘤、软组织和皮肤肿瘤、骨肿瘤等。In some preferred embodiments, 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.
在一些优选的实施方案中，神经系统肿瘤包括弥漫性胶质瘤，弥漫性星形细胞瘤和间变性星形细胞瘤，胶质母细胞瘤，少突胶质细胞瘤，少突星形细胞瘤，儿童弥漫性胶质瘤，其他星形细胞瘤，室管膜瘤，神经元和混合性神经元-神经胶质肿瘤，髓母细胞瘤，其他胚胎性肿瘤，神经鞘瘤，脑膜瘤，孤立性纤维性肿瘤和血管周细胞瘤等。In some preferred embodiments, 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.
在一些优选的实施方案中，头颈部肿瘤包括鼻腔及鼻窦恶性肿瘤，鼻咽癌，口腔癌，喉癌，涎腺肿瘤，颅内肿瘤，甲状腺癌，舌癌等。In some preferred embodiments, 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.
在一些优选的实施方案中，胸部肿瘤包括肺癌，食管癌，贲门癌，乳腺癌，纵膈肿瘤等。In some preferred embodiments, thoracic tumors include lung cancer, esophageal cancer, cardiac cancer, breast cancer, mediastinal tumors, and the like.
在一些优选的实施方案中，消化系统肿瘤包括胃癌，大肠癌及乙状结肠和直肠癌，肝癌，胰腺癌与壶腹周围癌，胆道癌，小肠恶性肿瘤等。In some preferred embodiments, digestive system tumors include gastric cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, small intestinal malignant tumors, and the like.
在一些优选的实施方案中，泌尿生殖系统肿瘤包括肾癌，前列腺癌，膀胱癌，睾丸恶性肿瘤，阴茎癌，子宫颈癌，子宫内膜癌，卵巢癌等。In some preferred embodiments, genitourinary tumors include kidney cancer, prostate cancer, bladder cancer, testicular malignancy, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, and the like.
在一些优选的实施方案中，软组织和皮肤肿瘤包括恶性纤维组织细胞瘤，横纹肌肉瘤，滑膜肉瘤，皮肤恶性黑色素瘤等。In some preferred embodiments, soft tissue and skin tumors include malignant fibrous histiocytoma, rhabdomyosarcoma, synovial sarcoma, cutaneous malignant melanoma, and the like.
在一些优选的实施方案中，骨肿瘤包括骨肉瘤，尤文氏肉瘤等。In some preferred embodiments, bone tumors include osteosarcoma, Ewing's sarcoma, and the like.
在一些优选的实施方案中，所述结肠癌是结肠腺瘤。In some preferred embodiments, the colon cancer is a colon adenoma.
在一些优选的实施方案中，所述乳腺癌是三阴乳腺癌细胞。In some preferred embodiments, the breast cancer is triple negative breast cancer cells.
在一些优选的实施方案中，所述肝癌是肝细胞癌。In some preferred embodiments, the liver cancer is hepatocellular carcinoma.
在一些优选的实施方案中，所述疾病是血液肿瘤选自白血病、淋巴瘤(HL)、多发性骨髓瘤(MM)、骨髓增生异常综合症(MDS)等In some preferred embodiments, the disease is a hematological neoplasm selected from the group consisting of leukemia, lymphoma (HL), multiple myeloma (MM), myelodysplastic syndrome (MDS), etc.
在一些优选的实施方案中，所述白血病是B细胞急性淋巴性白血病，T细胞急性淋巴性白血病，急性髓系白血病等。In some preferred embodiments, the leukemia is B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute myeloid leukemia, etc.
在另一方面，本公开还提供了一种治疗有需要的受试者的肿瘤或癌症的方法，包括：向所述受试者施用治疗有效量的本申请所述的溶瘤病毒或溶瘤病毒药物组合物。本发明的溶瘤病毒或组合物可向对象中单一剂量或多剂量施用。如果多剂量施用，可通过相同的或不同的路径进行施用，并且可在同一部位或不同部位施用。也可连续循环施用，在休息期之后重复。每次施用之间的间隔可从几小时到一年(例如24h、48h、72h、每周、每两周、每月或每年)。间隔也可是不定期的(例如在肿瘤发展之后)。每次施用剂量可在上述范围内变化。在某些实施方式中，施用溶瘤病毒的路径可以包括静脉内的和瘤内的路径。In another aspect, 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. In certain embodiments, the route of administration of the oncolytic virus may include intravenous and intratumoral routes.
在本发明的情况下，可以以合适的剂量(例如107到5x109pfu)一次或多次施用溶瘤病毒。每次病毒施用的间隔可从约1天到约8周范围内变化。另一优选治疗方案涉及IO8或IO9Pfu的溶瘤痘苗病毒的2到5次(例如3)静脉或瘤内施用，大约有1或2周间隔，其可以由医生根据实际情况选择。在一些实施方案中，所述方法包括向所述受试者施用包含如上文所述的核酸分子的溶瘤病毒载体，其中所述受试者患有癌症。所述药物组合物或包含如上文所述的核酸分子的溶瘤病毒载体能够靶向性降解致病蛋白，例如，致癌蛋白、病毒蛋白、免疫相关蛋白等。In the context of the present invention, the oncolytic virus may be administered once or multiple times at 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. In some embodiments, 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.
在一些实施方案中，所述疾病是感染类疾病，所述嵌合蛋白构建体的靶向结构域包含特异性靶向感染类疾病相关的靶点。所述感染类疾病相关的靶点如本申请前文所述。In some embodiments, the disease is an infectious disease, and 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.
在一些优选的实施方案中，病毒感染性疾病包括：呼吸道病毒性疾病，胃肠道病毒性疾病，肝脏病毒性疾病，皮肤和黏膜病毒性疾病，眼病毒性疾病，中枢神经系统病毒性疾病，淋巴细胞性病毒性疾病，虫传病毒性疾病，慢病毒感染疾病等。In some preferred embodiments, 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.
在一些优选的实施方案中，呼吸道病毒性疾病包括鼻病毒、腺病毒、呼吸道合胞病毒、副流感病毒和冠状病毒等的感染；流行性感冒；流行性腮腺炎等。 In some preferred embodiments, respiratory viral diseases include infections with rhinovirus, adenovirus, respiratory syncytial virus, parainfluenza virus, coronavirus, and the like; influenza; mumps, and the like.
在一些优选的实施方案中，胃肠道病毒性疾病包括脊髓灰质炎；库克萨基病毒感染；ECHO病毒感染；病毒性胃肠炎：包括轮状病毒性胃肠炎、诺瓦克病毒性胃肠炎、腺病毒性胃肠炎、星状病毒性胃肠炎、冠状病毒性胃肠炎和杯状病毒性胃肠炎等。In some preferred embodiments, 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.
在一些优选的实施方案中，肝脏病毒性疾病包括甲型病毒性肝炎、乙型病毒性肝炎、丙型病毒性肝炎、丁型病毒性肝炎、戊型病毒性肝炎、EB病毒性肝炎和巨细胞病毒性肝炎等。In some preferred embodiments, 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.
在一些优选的实施方案中，皮肤和黏膜病毒性疾病包括麻疹、风疹、幼儿急疹、水痘及带状疱疹、天花、单纯疱疹病毒感染、狂犬病和口蹄疫等。In some preferred embodiments, 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.
在一些优选的实施方案中，眼病毒性疾病包括流行性角膜结膜炎、滤泡性结膜炎和疱疹性角膜结膜炎等。In some preferred embodiments, ocular viral diseases include epidemic keratoconjunctivitis, follicular conjunctivitis, herpetic keratoconjunctivitis, and the like.
在一些优选的实施方案中，中枢神经系统病毒性疾病包括流行性乙型脑炎、西方马脑炎、东方马脑炎、圣路易脑炎、委内瑞拉马脑炎、墨累山谷脑炎、加利福尼亚脑炎、森林脑炎和淋巴细胞脉络丛脑膜炎等。In some preferred embodiments, 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.
在一些优选的实施方案中，淋巴细胞性病毒性疾病包括传染性单核细胞增多症、巨细胞病毒感染和获得性免疫缺陷综合征等。In some preferred embodiments, lymphocytic viral diseases include infectious mononucleosis, cytomegalovirus infection, acquired immunodeficiency syndrome, and the like.
在一些优选的实施方案中，虫传病毒性疾病包括病毒性出血热：包括流行性出血热、黄热病、克里米亚－刚果出血热、裂谷热、阿根廷出血热、玻利维亚出血热、拉萨热、鄂木斯克出血热、马尔堡病和埃波拉出血热等；登革热和登革出血热；西尼罗热；科罗拉多蜱传热；白蛉热等In some preferred embodiments, the insect-borne viral diseases include viral hemorrhagic fevers: including epidemic hemorrhagic fever, yellow fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever, Omsk hemorrhagic fever, Marburg disease and Ebola hemorrhagic fever, etc.; Dengue fever and dengue hemorrhagic fever; West Nile fever; Colorado tick-borne fever; sand fly fever, etc.
优选地，慢病毒感染疾病包括亚急性硬化性全脑炎、库鲁病、进行性多灶性白质脑病和亚急性海绵样脑病(皮质纹状体脊髓变性)等。Preferably, lentiviral infectious diseases include subacute sclerosing panencephalitis, kuru disease, progressive multifocal leukoencephalopathy, subacute spongiform encephalopathy (corticostriatal spinal cord degeneration), and the like.
在一些实施方案中，所述疾病是自身免疫性疾病，所述嵌合蛋白构建体的靶向结构域包含特异性靶向自身抗原相关的靶点。所述自身抗原相关的靶点如本申请前文所述。In some embodiments, 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.
在一些优选的实施方案中，自身免疫性疾病包括器官特异性自身免疫病和系统性自身免疫病In some preferred embodiments, autoimmune diseases include organ-specific autoimmune diseases and systemic autoimmune diseases
优选地，器官特异性自身免疫病包括慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症、急性特发性多神经炎等。Preferably, 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.
在一些优选的实施方案中，系统性自身免疫病包括系统性红斑狼疮、类风湿关节炎、系统性血管炎、硬皮病、天疱疮、皮肌炎、混合性结缔组织病、自身免疫性溶血性贫血、甲状腺自身免疫病、溃疡性结肠炎等。In some preferred embodiments, systemic autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune Hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, etc.
在一些实施方案中，所述疾病是神经类或退行性疾病，所述嵌合蛋白构建体的靶向结构域包含特异性靶向神经系统疾病相关的靶点。所述神经系统疾病相关的靶点如本申请前文所述。In some embodiments, the disease is a neurological or degenerative disease, and 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.
在一些实施方案中，所述神经类疾病包括神经系统疾病周围神经疾病如三叉神经痛、面瘫、面肌痉挛、前庭神经元炎、舌咽神经痛、单神经疾病、臂丛神经痛、多数性单神经病、多发性神经病、急性炎症性脱髓鞘性多发性神经病、慢性炎症性脱髓鞘性多发性神经病；In some embodiments, 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;
脊髓疾病如脊髓炎、压迫性脊髓病、脊髓亚急性联合变性、脊髓空洞症、脊髓血管病、脊髓蛛网膜炎等；Spinal cord diseases such as myelitis, compressive myelopathy, subacute combined degeneration of the spinal cord, syringomyelia, spinal vasculopathy, spinal arachnoiditis, etc.;
脑血管疾病如短暂性脑缺血发作、脑梗死、脑出血、蛛网膜下腔出血、颅内静脉系统血栓形成等；Cerebrovascular diseases such as transient ischemic attack, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, intracranial venous system thrombosis, etc.;
中枢神经系统感染性疾病如有病毒、细菌、真菌或寄生虫等感染导致的脑膜炎、脑炎等以及有慢病毒感染导致的慢病毒脑炎等；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;
中枢神经系统脱髓鞘性疾病如多发性硬化、视神经脊髓炎、急性传播性脑脊髓炎、脑白质营养不良等；Demyelinating diseases of the central nervous system such as multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, leukodystrophy, etc.;
运动障碍疾病如帕金森病、舞蹈病、肝豆状核变性、肌张力障碍、特发性震颤、迟发型运动障碍等；Movement disorders such as Parkinson's disease, chorea, hepatolenticular degeneration, dystonia, essential tremor, tardive dyskinesia, etc.;
癫痫；epilepsy;
头痛如偏头痛、紧张性头痛、丛集性头痛等；Headaches such as migraines, tension headaches, cluster headaches, etc.;
神经系统变性疾病如运动神经元病、阿尔茨海默病、路易体痴呆、额颞叶痴呆、多系统萎缩等；Nervous system degenerative diseases such as motor neuron disease, Alzheimer's disease, Lewy body dementia, frontotemporal dementia, multiple system atrophy, etc.;
神经系统遗传性疾病如遗传性共济失调、遗传性痉挛性截瘫、腓骨肌萎缩症、神经纤维瘤病、结节性硬化、脑面血管瘤病等；Genetic diseases of the nervous system such as hereditary ataxia, hereditary spastic paraplegia, peroneal muscular atrophy, neurofibromatosis, tuberous sclerosis, cerebrofacial angiomatosis, etc.;
神经系统发育异常性疾病如先天性脑积水、脑性瘫痪、颅底凹陷症、小脑扁桃体下病等；Nervous system developmental abnormalities such as congenital hydrocephalus, cerebral palsy, skull base depression, cerebellar subtonsillar disease, etc.;
神经肌肉和肌肉疾病如重症肌无力、周期性瘫痪、多发性肌炎、进行性肌营养不良症、肌强直性肌病(强直性肌营养不良症、先天性肌强直症)、代谢性肌病(线粒体肌病、脂质沉积性肌病、糖原沉积症)等；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.;
自主神经系统疾病如雷诺氏病、红斑性肢痛症、面偏侧萎缩症、全身自主神经功能不全、自发性多汗症、进行性脂肪营养不良等；Autonomic nervous system diseases such as Raynaud's disease, erythromelalgia, facial hemiatrophy, systemic autonomic insufficiency, spontaneous hyperhidrosis, progressive lipodystrophy, etc.;
神经系统肿瘤如脑胶质瘤、淋巴瘤、脊膜瘤等：Nervous system tumors such as glioma, lymphoma, meningioma, etc.:
神经系统副肿瘤综合症如副肿瘤性小脑变性、副肿瘤性脑脊髓炎、亚急性坏死性脊髓病、亚急性运动神经元病、副肿瘤性感觉神经元病等。Nervous system paraneoplastic syndromes such as paraneoplastic cerebellar degeneration, paraneoplastic encephalomyelitis, subacute necrotizing myelopathy, subacute motor neuron disease, paraneoplastic sensory neuron disease, etc.
在一些优选的实施方案中，神经退行性疾病会导致神经元结构和功能逐渐丧失，包括神经元死亡和胶质细胞平衡，会导致痴呆等认知障碍。示例性的神经退行性疾病包括：阿尔兹海默症、帕金森病(PD)、亨廷顿氏舞蹈病、早发性AD或PD、和肌萎缩性脊髓侧索硬化症(ALS)等。In some preferred embodiments, 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.
在一些实施方案中，所述疾病是代谢性疾病，例如糖尿病，高脂血症，痛风等。所述代谢性疾病相关的靶点如本申请前文所述。In some embodiments, the disease is a metabolic disease, such as diabetes, hyperlipidemia, gout, etc. The metabolic disease-related targets are as described previously in this application.
在一些实施方案中，前述LNP中的治疗性核酸的载体是转座子DNA。 In some embodiments, the vector for the therapeutic nucleic acid in the aforementioned LNP is transposon DNA.
在一个优选地实施方案中，所述LNP中可包载化疗药物，化疗药物包括但不限于细胞毒素、烷化剂、鬼臼类、喜树碱类、紫杉类、抗代谢类、抗生素类抗肿瘤药物中的一种或多种，可以列举的实例包括但不限于咪唑四嗪酮类药物，例如替莫唑胺；铂类药物，例如奥沙利铂、顺铂、卡铂、奈达铂、双环铂、乐铂、四硝酸三铂、菲铂、吡铂、沙铂；亚硝脲类，例如卡氮芥、环已亚硝脲、甲环亚硝脲、嘧啶亚硝脲、卡莫司汀、洛莫司汀、福莫司汀、尼莫司汀、雷莫司汀、链脲佐菌素；喜树碱类，例如喜树碱、羟基喜树碱、伊立替康、拓扑替康；长春碱类，例如长春瑞滨、长春碱、长春新碱、长春地辛、长春富宁；丙卡巴肼；盐酸米托蒽醌；氮芥类，例如氮芥、氮甲、环磷酰胺、异环磷酰胺、雌二醇氮芥、曲磷胺、苯丙氨酸氮芥、苯丁酸氮芥、美法仑、泼尼莫司汀、苯达莫司汀、乌拉莫司汀、雌莫司汀、噻替派；烷基磺酸酯类，例如白消安、甘露舒凡、苏消安；氟嘧啶衍生物，例如吉西他滨、卡培他滨、氟尿嘧啶、双呋氟尿嘧啶、去氧氟尿苷、替加氟、卡莫氟、三氟尿苷；紫杉烷类，例如紫杉醇、白蛋白结合的紫杉醇、紫杉醇脂质体以及多西他赛；培美曲塞、依托泊苷、伊立替康、丝裂霉素、阿糖胞苷、阿扎胞苷、氨柔比星、甲氨蝶呤、表阿霉素、阿霉素、Sapacitabine、普那布林、曲奥舒凡、地匹福林盐酸盐、替吉奥和encequidar中的一种或两种或三种。In a preferred embodiment, the LNP can contain chemotherapeutic drugs, which include but are not limited to cytotoxins, alkylating agents, podophyllotoxins, camptothecins, taxanes, antimetabolites, and antibiotics. One or more anti-tumor drugs, examples that can be cited 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; camptothecins, such as camptothecin, hydroxycamptothecin, irinotecan, topotecan; Vinblastines, such as vinorelbine, vinblastine, vincristine, vindesine, vinblastine; procarbazine; mitoxantrone hydrochloride; nitrogen mustards, such as nitrogen mustard, nitrogen mustard, cyclophosphamide, Cyclophosphamide, chlorambucil, chlorambucil, chlorambucil, melphalan, prednimustine, bendamustine, uramustine, estramustine Stin, thiotepa; alkyl sulfonate esters, such as busulfan, mannosulfan, sulfan; fluoropyrimidine derivatives, such as gemcitabine, capecitabine, fluorouracil, furfururacil, deoxyfluorouracil glycosides, tegafur, carmofur, trifluridine; taxanes such as paclitaxel, albumin-bound paclitaxel, paclitaxel liposomes, and docetaxel; pemetrexed, etoposide, iritidine Kang, Mitomycin, Cytarabine, Azacitidine, Amrubicin, Methotrexate, Epirubicin, Adriamycin, Sapacitabine, Plinabulin, Trioxovan, Dipi One or two or three of Folin hydrochloride, Tegio and encequidar.
在一个优选地实施方案中，所述LNP中可包载细胞毒素，所述细胞毒素包括但不限于海兔毒素(dolastatin)及奥瑞他汀(auristatin)类细胞毒分子，美登素(maytansine)类细胞毒分子；所述DNA损伤剂包括但不限于卡奇霉素类(calicheamicin)、倍癌霉素(duocarmycin)类、安曲霉素类衍生物PBD、喜树碱类衍生物SN-38。In a preferred embodiment, 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 .
更优选地，所述LNP中包载的细胞毒素选自瓢菌素(amanitins)、蒽环类物(anthracyclines)、浆果赤霉素(baccatins)、喜树碱(camptothecins)、西马多丁(cemadotins)、秋水仙碱(colchicines)、秋水仙胺(colcimids)、考布他汀(combretastatins)、隐菲辛(cryptophycins)、圆皮海绵内酯(discodermolides)、多烯紫杉醇(docetaxel)、阿霉素(doxorubicin)、棘霉素(echinomycins)、艾榴塞洛素(eleutherobins)、埃博霉素(epothilones)、雌莫司汀(estramustines)、偏端霉素(lexitropsins)、美登素(maytansines)、氨甲蝶呤(methotrexate)、纺锤菌素(netropsins)、嘌呤霉素(puromycins)、根瘤菌素(rhizoxins)、紫杉烷(taxanes)、微管蛋白裂解素(tubulysins)、或长春花生物碱(vinca alkaloids)。通过LNP递送融合蛋白的具体过程如图6所示(参见Lee NY,Ko WC,Hsueh PR.Nanoparticles in the Treatment of Infections Caused by Multidrug-Resistant Organisms.Front Pharmacol.2019 Oct 4；10:1153.doi:10.3389/fphar.2019.01153.PMID:31636564；PMCID:PMC6787836.)。More preferably, 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 specific process of delivering fusion proteins through LNP is shown in Figure 6 (see Lee NY, Ko WC, Hsueh PR. Nanoparticles in the Treatment of Infections Caused by Multidrug-Resistant Organisms. Front Pharmacol. 2019 Oct 4; 10:1153.doi: 10.3389/fphar.2019.01153.PMID:31636564; PMCID:PMC6787836.).
在一些实施方式中，前述融合蛋白与细胞毒素通过接头单元连接，所述细胞毒素包括但不限于海兔毒素(dolastatin)及奥瑞他汀(auristatin)类细胞毒分子，美登素(maytansine)类细胞毒分子；所述DNA损伤剂包括但不限于卡奇霉素类(calicheamicin)、倍癌霉素(duocarmycin)类、安曲霉素类衍生物PBD、喜树碱类衍生物SN-38。In some embodiments, 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.
更优选地，所述细胞毒素选自瓢菌素(amanitins)、蒽环类物(anthracyclines)、浆果赤霉素(baccatins)、喜树碱(camptothecins)、西马多丁(cemadotins)、秋水仙碱(colchicines)、秋水仙胺(colcimids)、考布他汀(combretastatins)、隐菲辛(cryptophycins)、圆皮海绵内酯(discodermolides)、多烯紫杉醇(docetaxel)、阿霉素(doxorubicin)、棘霉素(echinomycins)、艾榴塞洛素(eleutherobins)、埃博霉素(epothilones)、雌莫司汀(estramustines)、偏端霉素(lexitropsins)、美登素(maytansines)、氨甲蝶呤(methotrexate)、纺锤菌素(netropsins)、嘌呤霉素(puromycins)、根瘤菌素(rhizoxins)、紫杉烷(taxanes)、微管蛋白裂解素(tubulysins)、或长春花生物碱(vinca alkaloids)。More preferably, 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 .
在一些实施方式中，前述融合蛋白与标记连接，所述标记选自氧化铁纳米颗粒、紫外-可见标记、近红外标记、发光基团、磷光基团、磁自旋共振标记、光敏剂、光可裂解部分、螯合中心、重原子、放射性同位素、同位素可检测自旋共振标签，顺磁性部分，发色团或其组合；更优选地，所述标记为氧化铁纳米颗粒。In some embodiments, 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.
在一些实施方案中，前述融合蛋白、核酸、载体、细胞和/或前药可用于制备诊断、治疗或预防癌症或病毒感染类疾病的药物或试剂盒。In some embodiments, 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.
在一些实施方案中，前述疾病包括癌症和病毒感染类疾病。In some embodiments, the aforementioned diseases include cancer and viral infections.
在一些优选地实施方案中，前述癌症包括但不限于白血病、成人晚期癌症、胰腺癌、不可切除性胰腺癌、结肠直肠癌、转移性结肠直肠癌、卵巢癌、三阴性乳腺癌、造血/淋巴癌、结肠癌肝转移、小细胞肺癌、非小细胞肺癌、B细胞淋巴瘤、复发性或难治性B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、弥漫性大细胞淋巴瘤、复发性或难治性弥漫性大细胞淋巴瘤、间变性大细胞淋巴瘤、原发性纵隔B细胞淋巴瘤、复发性纵隔大B细胞淋巴瘤、难治性纵隔大B细胞淋巴瘤、大B细胞淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、复发性或难治性非霍奇金淋巴瘤、难治性侵袭性非霍奇金淋巴瘤、B细胞非霍奇金淋巴瘤、难治性非霍奇金淋巴瘤、结肠直肠上皮癌、胃癌、胰腺癌、三阴性浸润性乳腺癌、肾细胞癌、肺鳞状细胞癌、肝细胞癌、尿路上皮癌、白血病、B细胞白血病、B细胞急性淋巴细胞白血病、B细胞急性成淋巴细胞白血病、成人急性成淋巴细胞白血病、B细胞幼淋巴细胞白血病、儿童急性成淋巴细胞白血病、难治性儿童急性成淋巴细胞白血病、急性白血病、急性成淋巴细胞白血病、急性淋巴细胞白血病、幼淋巴细胞白血病、慢性淋巴细胞白血病、急性髓性白血病、复发性浆细胞骨髓瘤、难治性浆细胞骨髓瘤、多发性骨髓瘤、复发性或难治性多发性骨髓瘤、骨多发性骨髓瘤、脑恶性神经胶质瘤、骨髓增生异常综合征、EGFR阳性结肠直肠癌、多形性胶质母细胞瘤、赘生物、母细胞性浆细胞样树突状细胞肿瘤、肝转移、实体瘤、晚期实体瘤、间皮素阳性肿瘤、血液恶性肿瘤及其他晚期恶性肿瘤。In some preferred embodiments, 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, relapsed or refractory non-Hodgkin lymphoma, refractory aggressive non-Hodgkin lymphoma, B-cell non-Hodgkin lymphoma , refractory non-Hodgkin lymphoma, colorectal cancer, gastric cancer, pancreatic cancer, triple-negative invasive breast cancer, renal cell carcinoma, lung squamous cell carcinoma, hepatocellular carcinoma, urothelial carcinoma, leukemia, B cell leukemia, B-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, adult acute lymphoblastic leukemia, B-cell prolymphoblastic leukemia, childhood acute lymphoblastic leukemia, refractory childhood acute lymphoblastic leukemia, acute Leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, prolymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, relapsed plasma cell myeloma, refractory plasma cell myeloma, multiple myeloma, relapsed or refractory multiple myeloma, bone multiple myeloma, brain malignant glioma, myelodysplastic syndrome, EGFR-positive colorectal cancer, glioblastoma multiforme, neoplasm, blastic plasma Cytoid dendritic cell tumors, liver metastases, solid tumors, advanced solid tumors, mesothelin-positive tumors, hematological malignancies and other advanced malignancies.
在一些优选地实施方案中，所述病毒感染类疾病包括但不限于呼吸道病毒性疾病、胃肠道病毒性疾病、肝脏病毒性疾病、皮肤和黏膜病毒性疾病、眼病毒性疾病、中枢神经系统病毒性疾病、淋巴细胞性病毒性疾病、虫传病毒性疾病、慢病毒感染疾病等。In some preferred embodiments, 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.
在一些实施方案中，呼吸道病毒性疾病包括鼻病毒、腺病毒、呼吸道合胞病毒、副流感病毒和冠状病毒等的 感染；流行性感冒；流行性腮腺炎等；In some embodiments, respiratory viral diseases include rhinovirus, adenovirus, respiratory syncytial virus, parainfluenza virus, coronavirus, and the like. Infection; influenza; mumps, etc.;
更优选地，胃肠道病毒性疾病包括脊髓灰质炎；库克萨基病毒感染；ECHO病毒感染；病毒性胃肠炎：包括轮状病毒性胃肠炎、诺瓦克病毒性胃肠炎、腺病毒性胃肠炎、星状病毒性胃肠炎、冠状病毒性胃肠炎和杯状病毒性胃肠炎等。More preferably, 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.
在一些优选地实施方案中，肝脏病毒性疾病包括甲型病毒性肝炎、乙型病毒性肝炎、丙型病毒性肝炎、丁型病毒性肝炎、戊型病毒性肝炎、EB病毒性肝炎和巨细胞病毒性肝炎等。In some preferred embodiments, 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.
在一些优选地实施方案中，皮肤和黏膜病毒性疾病包括麻疹、风疹、幼儿急疹、水痘及带状疱疹、天花、单纯疱疹病毒感染、狂犬病和口蹄疫等。In some preferred embodiments, 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.
在一些优选地实施方案中，眼病毒性疾病包括流行性角膜结膜炎、滤泡性结膜炎和疱疹性角膜结膜炎等。In some preferred embodiments, ocular viral diseases include epidemic keratoconjunctivitis, follicular conjunctivitis, herpetic keratoconjunctivitis, and the like.
在一些优选地实施方案中，中枢神经系统病毒性疾病包括流行性乙型脑炎、西方马脑炎、东方马脑炎、圣路易脑炎、委内瑞拉马脑炎、墨累山谷脑炎、加利福尼亚脑炎、森林脑炎和淋巴细胞脉络丛脑膜炎等。In some preferred embodiments, 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.
在一些优选地实施方案中，淋巴细胞性病毒性疾病包括传染性单核细胞增多症、巨细胞病毒感染和获得性免疫缺陷综合征等。In some preferred embodiments, lymphocytic viral diseases include infectious mononucleosis, cytomegalovirus infection, acquired immunodeficiency syndrome, and the like.
在一些优选地实施方案中，虫传病毒性疾病包括病毒性出血热：包括流行性出血热、黄热病、克里米亚－刚果出血热、裂谷热、阿根廷出血热、玻利维亚出血热、拉萨热、鄂木斯克出血热、马尔堡病和埃波拉出血热等；登革热和登革出血热；西尼罗热；科罗拉多蜱传热；白蛉热等。In some preferred embodiments, the insect-borne viral diseases include viral hemorrhagic fevers: including epidemic hemorrhagic fever, yellow fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever, Omsk hemorrhagic fever, Marburg disease and Ebola hemorrhagic fever, etc.; Dengue fever and dengue hemorrhagic fever; West Nile fever; Colorado tick-borne fever; sand fly fever, etc.
在一些优选地实施方案中，慢病毒感染疾病包括亚急性硬化性全脑炎、库鲁病、进行性多灶性白质脑病和亚急性海绵样脑病(皮质纹状体脊髓变性)。In some preferred embodiments, lentiviral infectious diseases include subacute sclerosing panencephalitis, kuru disease, progressive multifocal leukoencephalopathy, and subacute spongiform encephalopathy (corticostriatal spinal degeneration).
在一些实施方案中，转座子是可以将本身插入基因组中的某位置处的一段DNA，例如，能够自我复制并将其副本插入基因组中的一段DNA或可以是从较长核酸剪接出来并插入基因组中的另一个位置的一段DNA。例如，转座子包含由反向重复序列组成的DNA序列，所述反向重复序列在用于换位的基因旁侧分布。In some embodiments, 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. For example, transposons comprise DNA sequences composed of inverted repeats that flank the gene for transposition.
术语“转座子”是指由转座酶或整合酶识别并且是能够进行转座的功能性核酸-蛋白质复合物(即，转座体)的基本组分的核酸区段。如本文所用，术语“转座酶”是指一种酶，它是能够进行转座的功能性核酸-蛋白质复合物的组分并介导转座。术语“转座酶”还指来自逆转录转座子或起源于逆转录病毒的整合酶。转座子复合物在转座酶和双链DNA片段之间形成，所述片段含有对该酶的特异性结合序列，称为“转座子末端”。转座子结合位点的序列可以在某些位置用其他碱基修饰，而不影响转座子复合物形成可以有效地转座成靶DNA的稳定结构的能力The term "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). As used herein, the term "transposase" refers to an enzyme that is a component of a functional nucleic acid-protein complex capable of transposition and mediates transposition. The term "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.
术语“化疗药物”是指任何对受试者具有治疗效果的化学药物。“化疗药物”包括但不限于抗肿瘤药，所述化疗药物包括但不限于海兔毒素(dolastatin)及奥瑞他汀(auristatin)类细胞毒分子，美登素(maytansine)类细胞毒分子；所述DNA损伤剂包括但不限于卡奇霉素类(calicheamicin)、倍癌霉素(duocarmycin)类、安曲霉素类衍生物PBD、喜树碱类衍生物SN-38、瓢菌素(amanitins)、蒽环类物(anthracyclines)、浆果赤霉素(baccatins)、喜树碱(camptothecins)、西马多丁(cemadotins)、秋水仙碱(colchicines)、秋水仙胺(colcimids)、考布他汀(combretastatins)、隐菲辛(cryptophycins)、圆皮海绵内酯(discodermolides)、多烯紫杉醇(docetaxel)、阿霉素(doxorubicin)、棘霉素(echinomycins)、艾榴塞洛素(eleutherobins)、埃博霉素(epothilones)、雌莫司汀(estramustines)、偏端霉素(lexitropsins)、美登素(maytansines)、氨甲蝶呤(methotrexate)、纺锤菌素(netropsins)、嘌呤霉素(puromycins)、根瘤菌素(rhizoxins)、紫杉烷(taxanes)、微管蛋白裂解素(tubulysins)、或长春花生物碱(vinca alkaloids)、烷化剂、鬼臼类、喜树碱类、紫杉类、抗代谢类、抗生素类抗肿瘤药物中的一种或多种，可以列举的实例包括但不限于咪唑四嗪酮类药物，例如替莫唑胺；铂类药物，例如奥沙利铂、顺铂、卡铂、奈达铂、双环铂、乐铂、四硝酸三铂、菲铂、吡铂、沙铂；亚硝脲类，例如卡氮芥、环已亚硝脲、甲环亚硝脲、嘧啶亚硝脲、卡莫司汀、洛莫司汀、福莫司汀、尼莫司汀、雷莫司汀、链脲佐菌素；喜树碱类，例如喜树碱、羟基喜树碱、伊立替康、拓扑替康；长春碱类，例如长春瑞滨、长春碱、长春新碱、长春地辛、长春富宁；丙卡巴肼；盐酸米托蒽醌；氮芥类，例如氮芥、氮甲、环磷酰胺、异环磷酰胺、雌二醇氮芥、曲磷胺、苯丙氨酸氮芥、苯丁酸氮芥、美法仑、泼尼莫司汀、苯达莫司汀、乌拉莫司汀、雌莫司汀、噻替派；烷基磺酸酯类，例如白消安、甘露舒凡、苏消安；氟嘧啶衍生物，例如吉西他滨、卡培他滨、氟尿嘧啶、双呋氟尿嘧啶、去氧氟尿苷、替加氟、卡莫氟、三氟尿苷；紫杉烷类，例如紫杉醇、白蛋白结合的紫杉醇、紫杉醇脂质体以及多西他赛；培美曲塞、依托泊苷、伊立替康、丝裂霉素、阿糖胞苷、阿扎胞苷、氨柔比星、甲氨蝶呤、表阿霉素、阿霉素、Sapacitabine、普那布林、曲奥舒凡、地匹福林盐酸盐、替吉奥和encequidar中的一种或两种或三种The term "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, maytansines, methotrexate, netropsins, puromycin puromycins), rhizoxins, taxanes, tubulysins, or vinca alkaloids, alkylating agents, podophyllins, camptothecins, purple One or more of the anti-tumor drugs of finanoids, antimetabolites, and antibiotics. 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; mitoxantrone hydrochloride; nitrogen mustards, such as nitrogen mustard , chlorambucil, cyclophosphamide, ifosfamide, estradiol mustard, traphosfamide, phenylalanine mustard, chlorambucil, melphalan, prednimustine, bendambucil Stin, uramustine, estramustine, thiotepa; alkyl sulfonate esters, such as busulfan, mannosulfan, sulfan; fluoropyrimidine derivatives, such as gemcitabine, capecitabine, fluorouracil , difurfururacil, deoxyfluridine, tegafur, carmofur, trifluridine; taxanes, such as paclitaxel, albumin-bound paclitaxel, paclitaxel liposomes, and docetaxel; Pemet Trecet, etoposide, irinotecan, mitomycin, cytarabine, azacitidine, amrubicin, methotrexate, epirubicin, doxorubicin, sapacitabine, pranabine One, two, or three of lin, troxofan, dipifrine hydrochloride, tigio and encequidar
术语“细胞毒素”包括任何能杀伤T细胞或降低其活性的化合物(例如药物)，所述细胞毒素包括但不限于海兔毒素(dolastatin)及奥瑞他汀(auristatin)类细胞毒分子，美登素(maytansine)类细胞毒分子；所述DNA损伤剂包括但不限于卡奇霉素类(calicheamicin)、倍癌霉素(duocarmycin)类、安曲霉素类衍生物PBD、喜树碱类衍生物SN-38、瓢菌素(amanitins)、蒽环类物(anthracyclines)、浆果赤霉素(baccatins)、喜树碱(camptothecins)、西马多丁(cemadotins)、秋水仙碱(colchicines)、秋水仙胺(colcimids)、考布他汀(combretastatins)、隐菲辛(cryptophycins)、圆皮海绵内酯(discodermolides)、多烯紫杉醇(docetaxel)、阿霉素(doxorubicin)、棘霉素(echinomycins)、艾榴塞洛素(eleutherobins)、埃博霉素(epothilones)、雌莫司汀(estramustines)、偏端霉素(lexitropsins)、美登素(maytansines)、氨甲蝶呤(methotrexate)、纺锤菌素(netropsins)、嘌呤霉素(puromycins)、根瘤菌素(rhizoxins)、紫杉烷(taxanes)、微管蛋白裂解素(tubulysins)、或长春花生物碱(vinca alkaloids)。 The term "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, taxanes, tubulysins, or vinca alkaloids.
术语“接头”是指包含一个或多个氨基酸通常约2-20个氨基酸的肽。肽接头在本领域中是已知的或描述于本文中。适合的非免疫原性接头肽例如是，(G4S)n、(SG4)n或G4(SG4)n肽接头，其中“n”通常为1至10之间的数字，通常为1至4，特别是2。在本文中出现的“接头L1”包括但不限于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.29)、GGSGSGSG(SEQ ID NO.30)、GGSGSG(SEQ ID NO.31)、GGSG(SEQ ID NO.32)。在本文中出现的“接头L2”可以是在前述“接头L1”的C末端加入了一个半胱氨酸残基的多肽，包括但不限于GGGSC(SEQ ID NO.33)、GGGGSGGGGSC(SEQ ID NO.34)、GGGGSGGGGSGGGGSGGGGSC(SEQ ID NO.35)和GGGGSGGGGSGGGSGGGGSC(SEQ ID NO.36)。The term "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. The "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). 29), GGSGSGSG(SEQ ID NO.30), GGSGSG(SEQ ID NO.31), GGSG(SEQ ID NO.32). The "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).
术语“接头单元”是指一端与配体共价连接而另一端与细胞毒性药物相连的化学结构片段或键。本文中所使用的接头单元可包括但不限于：马来酰亚胺基-己酰基-缬氨酸-瓜氨酸-p-氨基苄氧基(Maleimido-Caproyl-Valine-Citrulline-p-Aminobenzyloxy,mc-vc-pAB)、马来酰亚胺基己酰基(Maleimidocaproyl，mc)、三甘氨酸肽接头(triglycyl peptide linker)、3-马来酰亚胺-丙酸(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-pyrrol-1-yl)ethoxy)ethoxy)propanoate)、Mal-Tri-EG-OSu(2,5-dioxopyrrolidin-1-yl 3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxypro panoate)、Mal-Tetra-EG-OSu(2,5-dioxopyrrolidin-1-yl 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16-tetraoxa-4-azan onadecan-19-oate)、Br-di-EG-OSu(2,5-dioxopyrrolidin-1-yl 3(2-(2-(2-bromoacetamido)ethoxy)ethoxy)propanoate)、Py-ds-prp-OSu(2-5-dioxopyrrolidin-1-yl 3-(pyridine-2-yldisulfanyl)propanoate)、Py-ds-Prp-OPEP(perfluorophenyl 3-(pyridine-2-yldisulfanyl)propanoate)、Py-ds-dmBut-OSu(2,5-dioxopyrrolidin-1-yl 4-methyl-4-(pyridine-2-yldisulfanyl)、Py-ds-dmBut-OPF(perfluorophenyl 4-methyl-4-(pyridine-2-yldisulfanyl)pentanoate)、SMCC(N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate)、MBS(3-maleimidobenzoic acid N-hydroxysuccinimide ester)、SATA(S-(N-succinimidyl)thioacetate)、SPDP((N-succinimidyl 3-(2-pyridyldithio)propionate)、SMPT((N-succinimidyloxy carbonyl)-1-methyl-1-(2-pyridyldithio)toluene)。The term "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-pyrrol-1-yl)ethoxy)ethoxy) propanoate), Mal-Tri-EG-OSu(2,5-dioxopyrrolidin-1-yl 3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)ethoxy)ethoxy)ethoxypro panoate), Mal-Tetra-EG-OSu(2,5-dioxopyrrolidin-1-yl 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -3-oxo-7,10,13,16-tetraoxa-4-azan onadecan-19-oate), Br-di-EG-OSu(2,5-dioxopyrrolidin-1-yl 3(2-(2-( 2-bromoacetamido)ethoxy)ethoxy)propanoate), Py-ds-prp-OSu(2-5-dioxopyrrolidin-1-yl 3-(pyridine-2-yldisulfanyl)propanoate), Py-ds-Prp-OPEP(perfluorophenyl 3 -(pyridine-2-yldisulfanyl)propanoate), Py-ds-dmBut-OSu(2,5-dioxopyrrolidin-1-yl 4-methyl-4-(pyridine-2-yldisulfanyl), Py-ds-dmBut-OPF( perfluorophenyl 4-methyl-4-(pyridine-2-yldisulfanyl)pentanoate), SMCC(N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate), MBS(3-maleimidobenzoic acid N-hydroxysuccinimide ester), SATA(S-(N-succinimidyl )thioacetate), SPDP ((N-succinimidyl 3-(2-pyridyldithio)propionate), SMPT ((N-succinimidyloxy carbonyl)-1-methyl-1-(2-pyridyldithio)toluene).
术语“标记”在本文中用来指这样的化合物或组合物，其直接或间接地与例如核酸探针或抗体等试剂偶联或融合，并有助于检测与其偶联或融合的所述试剂。标记本身可以是可检测的(例如放射性同位素标记或荧光标记)，或者，对酶标记而言，可催化可检测的底物反应物或组合物的化学变化。标记可包括氧化铁纳米颗粒、紫外-可见标记、近红外标记、发光基团、磷光基团、磁自旋共振标记、光敏剂、光可裂解部分、螯合中心、重原子、放射性同位素、同位素可检测自旋共振标签、顺磁性部分、发色团或其组合。The term "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.
术语“药物制剂”指形式为允许其中所含活性成分的生物活性有效，并且不包含对将要施用该制剂的受试者有不可接受的毒性的另外组分的制备物。The term "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.
术语“药学上可接受的载体”指药物制剂中除活性成分之外的成分，其对受试者无毒。药学上可接受的载体包括但不限于缓冲剂、赋形剂、稳定剂或防腐剂。The term "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.
在一些方面，载体的选择部分地由特定细胞和/或施用方法来确定。因此，存在多种合适的制剂。例如，药物组合物可包含防腐剂。合适的防腐剂可包括例如对羟基苯甲酸甲酯、对羟基苯甲酸丙酯、苯甲酸钠和苯扎氯铵。在一些方面，使用两种或更多种防腐剂的混合物。防腐剂或其混合物通常以组合物总重量的约0.0001重量％至约2重量％的量存在。药学上可接受的载体通常在使用的剂量和浓度下对受体无毒，并包括但不限于：缓冲剂，例如磷酸盐、柠檬酸盐和其他有机酸；抗氧化剂，包括抗坏血酸和蛋氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；氯化六甲双铵(hexamethonium chloride)；苯扎氯铵；苄索氯铵；苯酚、丁醇或苄醇；对羟基苯甲酸烷基酯，例如对羟基苯甲酸甲酯或对羟基苯甲酸丙酯；邻苯二酚；间苯二酚；环己醇；3-戊醇和间甲酚)；低分子量(少于约10个残基)多肽；蛋白质，例如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物，例如聚乙烯吡咯烷酮；氨基酸，例如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖和其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂，如EDTA；糖，例如蔗糖、甘露醇、海藻糖或山梨糖醇；盐形成抗衡离子，例如钠；金属络合物(例如锌蛋白络合物)；和/或非离子表面活性剂，例如聚乙二醇(PEG)。In some aspects, the choice of vector is determined in part by the specific cells and/or method of administration. Therefore, a variety of suitable formulations exist. For example, 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, asparagine, histidine, arginine or lysine Acids; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salts that form counterions, such as sodium; Metal complexes (eg zinc protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).
在一些方面，缓冲剂包括在组合物中。合适的缓冲剂包括例如柠檬酸、柠檬酸钠、磷酸、磷酸钾和各种其他酸和盐。在一些方面，使用两种或更多种缓冲剂的混合物。缓冲剂或其混合物通常以组合物总重量的约0.001重量％至约4重量％的量存在。制备可施用的药物组合物的方法是已知的。In some aspects, 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. In some aspects, 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.
制剂可以包括水溶液。制剂或组合物还可包含多于一种可用于正在用细胞治疗的特定适应症、疾病或病症的活性成分，优选具有与细胞互补的活性的那些，其中各自的活性不会彼此不利地影响。这样的活性成分合适地以对于预期目的有效的量组合存在。因此，在一些实施方式中，药物组合物还包含其他药物活性剂或药物，例如化学治疗剂例如天冬酰胺酶、白消安、卡铂、顺铂、柔红霉素、阿霉素、氟尿嘧啶、吉西他滨、羟基脲、甲氨蝶呤、紫杉醇、利妥昔单抗、长春碱和/或长春新碱。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. Such active ingredients are suitably present in combination in amounts effective for the intended purpose. Thus, in some embodiments, 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.
在一些实施方式中，药物组合物包含有效治疗或预防疾病或病症的量的细胞，例如治疗有效量或预防有效量。在一些实施方式中，通过定期评估所治疗的受试者来监测治疗或预防功效。可以通过单次推注施用细胞、多次推注施用细胞，或连续输注施用细胞来递送期望剂量。 In some embodiments, 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. In some embodiments, 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.
细胞和组合物可以使用标准施用技术、制剂和/或装置来施用。细胞的施用可以是自体的或异源的。例如免疫应答细胞或祖细胞可以从一个受试者获得，并施用于相同受试者或不同的、相容的受试者。外周血衍生的免疫应答细胞或其后代(例如体内、离体或体外衍生的)可以通过局部注射施用，包括导管施用、全身注射、局部注射、静脉注射或肠胃外施用。当施用治疗组合物(例如包含遗传修饰的免疫应答细胞的药物组合物)时，通常将其配制为单位剂量可注射形式(溶液、悬浮液、乳剂)。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).
制剂包括用于口服、静脉内、腹膜内、皮下、肺、透皮、肌内、鼻内、颊、舌下或栓剂施用的那些。在一些实施方式中，细胞群是肠胃外施用的。如在本文中所使用的术语“肠胃外”包括静脉内、肌内、皮下、直肠、阴道和腹膜内施用。在一些实施方式中，细胞通过静脉内，腹膜内或皮下注射施用使用外周全身递送而施用至受试者。Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell population is administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal and intraperitoneal administration. In some embodiments, cells are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
在一些实施方式中，组合物作为无菌液体制备物提供，例如等渗水溶液、悬浮液、乳剂、分散液或粘性组合物，其在某些方面可以缓冲至选定的pH。液体制备物通常比凝胶、其他粘性组合物和固体组合物更容易制备。另外，液体组合物在某种程度上更方便施用，尤其是通过注射。另一方面，粘性组合物可以在适当的粘度范围内配制以提供与特定组织更长的接触时间。液体或粘性组合物可包含载体，其可以是溶剂或分散介质，包含例如水、盐水、磷酸盐缓冲盐水、多元醇(例如甘油、丙二醇、液体聚乙二醇)及其合适的混合物。In some embodiments, 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. 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.
无菌可注射溶液可以通过将细胞引入溶剂中来制备，例如与合适的载体、稀释剂或赋形剂如无菌水、生理盐水、葡萄糖、右旋糖等混合。组合物可以包含辅助物质，例如润湿剂、分散剂或乳化剂(例如甲基纤维素)、pH缓冲剂、胶凝或增粘添加剂、防腐剂、矫味剂和/或颜色，这取决于施用途径和期望制备物。在某些方面，可以参考标准文本以制备合适的制备物。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. 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. In some respects, reference can be made to standard texts for the preparation of suitable preparations.
可以添加增强组合物的稳定性和无菌性的各种添加剂，包括抗微生物防腐剂、抗氧化剂、螯合剂和缓冲剂。微生物作用的预防可以通过各种抗细菌和抗真菌剂来确保，例如对羟基苯甲酸酯、氯丁醇、苯酚和山梨酸。可注射药物形式的延长吸收可通过使用延迟吸收的试剂例如单硬脂酸铝和明胶来实现。Various 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.
用于体内施用的制剂通常是无菌的。无菌性可以通过例如经过无菌滤膜过滤而容易地实现。Formulations for in vivo administration are generally sterile. Sterility can be easily achieved by, for example, filtration through a sterile membrane.
还提供了施用细胞、群体和组合物以治疗或预防疾病，病症和紊乱(包括癌症)的方法，以及这样的细胞、群体和组合物治疗或预防疾病，病症和紊乱(包括癌症)的用途。在一些实施方式中，将细胞、群体和组合物施用于患有特定疾病或病症的待治疗受试者或患者，例如通过过继细胞疗法(例如过继T细胞疗法)。在一些实施方式中，将通过提供的方法制备的细胞和组合物(例如工程化组合物和孵育和/或其他加工步骤后的生产末端产物)施用于受试者，例如患有疾病或病症或处于疾病或病症风险的受试者。在一些方面，方法由此治疗例如疾病或病症的一种或多种症状，例如通过减轻表达被工程化T细胞识别的抗原的癌症中的肿瘤负担。Also provided are methods of administering cells, populations and compositions to treat or prevent diseases, conditions and disorders (including cancer), as well as uses of such cells, populations and compositions to treat or prevent diseases, conditions and disorders (including cancer). In some embodiments, 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). In some embodiments, cells and compositions (e.g., engineered compositions and production end products after incubation and/or other processing steps) prepared by the provided methods are administered to a subject, e.g., suffering from a disease or disorder or Subjects at risk for disease or condition. In some aspects, 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.
用于过继细胞疗法的细胞施用方法是已知的，并且可以与所提供的方法和组合物结合使用。例如，过继性T细胞疗法的方法在例如Gruenberg等的美国专利申请公开号2003/0170238；Rosenberg的美国专利号4,690,915；Rosenberg(2011)Nat Rev Clin Oncol.8(10):577-85)中描述。参见例如Themeli等(2013)Nat Biotechnol.31(10):928-933；Tsukahara等(2013)Biochem Biophys Res Commun 438(1):84-9；Davila等(2013)PLoS ONE 8(4):e61338。Cell administration methods for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, methods of adoptive T cell therapy are 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) . See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10):928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9; Davila et al. (2013) PLoS ONE 8(4):e61338 .
如在本文中所使用的“受试者”是哺乳动物，例如人或其他动物，并且通常是人。在一些实施方式中，向其施用细胞、细胞群或组合物的受试者例如患者是哺乳动物，通常是灵长类动物例如人。在一些实施方式中，灵长类动物是猴子或猿。受试者可以是男性或女性，并且可以是任何合适的年龄，包括婴儿、少年、青少年、成人和老年受试者。在一些实施方式中，受试者是非灵长类哺乳动物，例如啮齿动物。A "subject" as used herein is a mammal, such as a human or other animal, and is typically a human. In some embodiments, 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. In some embodiments, 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. In some embodiments, the subject is a non-primate mammal, such as a rodent.
如在本文中所使用的“治疗”(及其语法变化，例如“治疗”或“治疗”)指完全或部分改善或减轻疾病或病症或紊乱，或与之相关的症状、不良作用或结果或表型。期望的治疗效果包括但不限于预防疾病的发生或复发、症状的减轻、疾病的任何直接或间接病理后果的减轻、预防转移、降低疾病进展速度、缓解或减轻疾病状态以及缓解或改善预后。这些术语并不暗示完全治愈疾病或完全消除任何症状或对所有症状或后果的影响。"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.
如在本文中所使用的“延缓疾病的发展”是指推迟、阻碍、减缓、阻滞、稳定、抑制和/或延迟疾病(例如癌症)的发展。该延缓可以具有不同的时间长度，这取决于疾病的历史和/或被治疗的个体。对本领域技术人员显而易见的是，足够或明显的延迟实际上可以包括预防，因为个体不发生该疾病。例如，晚期癌症例如转移的发展可能被延迟。As used herein, "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.
如在本文中所使用的“预防”包括针对可易患疾病但尚未被诊断患有该疾病的受试者中该疾病的发生或复发提供预防。在一些实施方式中，提供的细胞和组合物用于延迟疾病的发展或减慢疾病的进展。"Prevention" as used herein 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. In some embodiments, cells and compositions are provided for delaying the development of a disease or slowing the progression of a disease.
如在本文中所使用的“抑制”功能或活性是指当与相同条件(除感兴趣的条件或参数)相比时，或者与另外的条件相比时，降低功能或活性。例如，抑制肿瘤生长的细胞与在该细胞不存在下的肿瘤的生长速率相比降低肿瘤的生长速率。"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.
在施用的上下文中，试剂(例如嵌合抗原受体、多核苷酸、载体、药物制剂、细胞或组合物)的“有效量”指在必要的剂量/量和时间段下，有效达到期望结果例如治疗或预防结果的量。In the context of administration, an "effective amount" of an agent (e.g., chimeric antigen receptor, polynucleotide, vector, pharmaceutical formulation, cell or composition) means effective in achieving the desired result at the dose/amount and time period necessary For example, the amount of therapeutic or preventive results.
试剂(例如药物制剂或细胞)的“治疗有效量”指在必要的剂量和时间段下，有效达到期望治疗效果(例如用于治疗疾病、病症、或紊乱，和/或治疗的药代动力学或药效学作用)的量。治疗有效量可以根据例如疾病状态、受试者的年龄、性别和体重以及所施用的细胞群等因素而变化。在一些实施方式中，提供的方法包括以有效量例如治疗有效量施用细胞和/或组合物。 A "therapeutically effective amount" of an agent (e.g., a pharmaceutical preparation or a cell) 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. In some embodiments, provided methods include administering cells and/or compositions in an effective amount, such as a therapeutically effective amount.
“预防有效量”指在必要的剂量和时间段下，有效达到期望预防结果的量。通常但不是必须的，因为在疾病之前或早期在受试者中使用预防剂量，所以预防有效量将小于治疗有效量。在较低肿瘤负荷的情况下，在某些方面中预防有效量将高于治疗有效量。"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.
在某些实施方式中，将细胞或细胞的个体群体或亚型以约一百万至约一千亿个细胞的范围施用于受试者，例如100万至约500亿个细胞(例如约500万个细胞、约2500万个细胞、约5亿个细胞、约10亿个细胞、约50亿个细胞、约200亿个细胞、约300亿个细胞、约400亿个细胞、或任意上述两个值定义的范围)，例如约1000万到约1000亿个细胞(例如约2000万个细胞、约3000万个细胞、约4000万个细胞、约6000万个细胞、约7000万个细胞、约8000万个细胞、约9000万个细胞、约100亿个细胞、约250亿个细胞、约500亿个细胞、约750亿个细胞、约900亿个细胞，或任意上述两个值定义的范围)，和在某些情况下，约1亿个细胞至约500亿个细胞(例如，约1.2亿个细胞、约2.5亿个细胞、约3.5亿个细胞、约4.5亿个细胞、约6.5亿个细胞、约8亿个细胞、约9亿个细胞、约30亿个细胞、约300亿个细胞、约450亿个细胞)或这些范围之间的任何值。In certain embodiments, 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, about 30 billion cells, about 45 billion cells) or any value between these ranges.
在一些实施方式中，总细胞的剂量和/或细胞的个体亚群的剂量在处于或大约104个细胞/千克(kg)体重至处于或大约109个细胞/千克(kg)体重之间的范围内，例如在105和106个细胞/kg体重之间，例如，至少或至少大约或处于或大约1×105个细胞/kg、1.5×105个细胞/kg，2×105个细胞/kg或1×106个细胞/kg体重。例如，在一些实施方式中，细胞以在处于或大约104和处于或约109个T细胞/千克(kg)体重之间或其一定误差范围内施用，例如在105和106个T细胞/kg体重之间，例如至少或至少大约或处于或大约1×105个T细胞/kg、1.5×105个T细胞/kg，2×105个T细胞/kg、或1×106个T细胞/kg体重。In some embodiments, 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. For example, in some embodiments, 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.
细胞可以通过任何合适的方式来施用，例如通过推注，通过注射，例如静脉内或皮下注射、眼内注射、眼周注射、视网膜下注射、玻璃体内注射、经中隔注射、巩膜下注射、脉络膜内注射、前房内注射、会阴下注射、结膜下注射、眼球筋膜囊下(sub-Tenon)注射、球后注射、球周注射，或后近巩膜(posterior juxtascleral)递送。在一些实施方式中，它们通过肠胃外、肺内和鼻内施用，并且如果局部治疗是期望的，则通过病灶内施用。肠胃外输注包括肌内、静脉内、动脉内、腹膜内或皮下给药。在一些实施方式中，给定剂量通过单次推注施用细胞来施用。在一些实施方式中，其通过多次推注施用细胞来施用，例如，在不超过3天的期间，或通过连续输注施用细胞来施用。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. In some embodiments, 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. In some embodiments, 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.
在一些实施方式中，提供了重复剂量方法，其中给予第一剂量的细胞，然后给予一个或多个第二连续剂量。当以过继治疗方法施用于受试者时，通常设计细胞的多剂量的时机和大小以增加表达抗原的T细胞(例如表达CAR的T细胞)的功效和/或活性和/或功能。在一些实施方式中，重复给药减少了当抑制性免疫分子例如PD-1和/或PD-L1在表达抗原的例如表达CAR的T细胞上被上调时可发生的下调或抑制活性。方法包括施用第一剂量，通常随后是一个或多个连续剂量，并且在不同剂量之间具有特定的时间范围。In some embodiments, repeated dosing methods are provided wherein a first dose of cells is administered followed by one or more second consecutive doses. When administered to a subject in an adoptive therapy approach, 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). In some embodiments, 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.
在过继细胞疗法的背景下，给定“剂量”的施用包括作为单一组合物和/或单次不间断施用(例如，作为单次注射或连续输注)的给定量或数量的细胞的施用，并且还包括在指定的时间段(不超过3天)中，以多个个体组合物或输注提供的给定量或数量的细胞数量作为分次剂量的施用。因此，在某些情况下，第一或连续剂量是在单个时间点给予或启动的指定数量细胞的单次或连续施用。然而，在某些情况下，第一或连续剂量在不超过三天的时间段内以多次注射或输注施用，例如三天或两天、每天一次，或在一天时间内多次输注。In the context of adoptive cell therapy, administration of a given "dose" includes administration of a given amount or number of cells as a single composition and/or 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). Thus, in some cases, 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. However, in some cases, 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. .
在下面对本公开的实施例的详细描述中，参考了附图。在附图中相似的附图标记指示相似的元素，并且在附图中以说明的方式示出了可以实践本公开的特定实施方式。对这些实施方式进行了足够详细的描述，以使本领域技术人员能够实施本公开。在其他情况下，未详细示出公知的过程、结构和技术，以免混淆对本说明书的理解。因此，以下详细描述不应被理解为限制性的，并且本公开的技术方案仅由所附权利要求书限定。In the following detailed description of embodiments of the present disclosure, reference is made to the accompanying drawings. In the drawings, like reference numerals indicate similar elements, and there is shown by way of illustration specific embodiments in which the disclosure may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the disclosure. In other instances, well-known processes, structures and techniques have not been shown in detail so as not to obscure the understanding of this specification. Therefore, the following detailed description should not be construed as limiting, and the technical solution of the present disclosure is limited only by the appended claims.
实施例Example
实施例1：靶向抗原促进抗原内吞的融合蛋白Example 1: Fusion protein targeting antigen to promote antigen endocytosis
本实施例设计了一种促进靶向抗原内吞的融合蛋白，在此基础上，本实施例设计了靶向TCR促进TCR内吞的融合蛋白。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.
1.1靶向抗原促进抗原内吞的融合蛋白1.1 Fusion proteins that target antigens and promote antigen endocytosis
该融合蛋白的基本结构如图1A所示，其包括靶向结合结构域(Targeting Domain)，接头(Linker)，三聚体结构域(Trimer domain)。The basic structure of the fusion protein is shown in Figure 1A, which includes a targeting domain, a linker, and a trimer domain.
在一些实施方式中，该融合蛋白的蛋白结构形式可能为单体，二聚体(靶向结构域形成异二聚体)，二聚体(靶向结构域形成同二聚体)或三聚体。如图1G所示。In some embodiments, 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.
1.2靶向TCR促进TCR内吞的融合蛋白1.2 Fusion proteins targeting TCR to promote TCR endocytosis
在一些实施方式中，本实施例设计了靶向TCR促进TCR内吞的融合蛋白BMA-STII-VXT(该融合蛋白BMA-STII-VXT的氨基酸序列如SEQ ID NO.36所示)，其包括靶向TCR的单链抗体片段(scFv)，Strep tag II标签，甘丝氨酸铰链区(Glycine-Serine linker)和来自胶原蛋白XV的三聚体结构域(VXT)，如图1B所示。In some embodiments, 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.
其中靶向TCRαβ的单链抗体为特异性结合TCRαβ/CD3复合物恒定表位的抗体BMA031：The single-chain antibody targeting TCRαβ is the antibody BMA031 that specifically binds to the constant epitope of the TCRαβ/CD3 complex:
靶向TCR的单链抗体BMA031的重链可变区的氨基酸序列如SEQ ID NO.7所示

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

其中，STII(Trp-Ser-His-Pro-Gln-Phe-Glu-Lys)是8个氨基酸的Strep Tag II标签，Strep-tag II对Strep-Tactin的结合亲和力比对streptavidin高将近100倍，因此可以用Strep-Tactin进行纯化；VXT是促进三聚体形成的组件，其来自人源XV型胶原蛋白的三聚体结构域(VXT的氨基酸序列如SEQ ID NO.18所示)。VXT结构域包含4个β-折叠结构和α-螺旋，可以形成三螺旋的结构。Among them, STII (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) is an 8-amino-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 β-sheet structures and α-helices, which can form a triple-helix structure.
在一些实施方案中，将编码靶向TCR促进TCR内吞的融合蛋白的核酸序列克隆到真核表达质粒中，将质粒转染工程细胞系Expi293(购自ThermoFisher，货号:A14635)培养7天后，收集培养基上清液，经过使用纯化柱Starm Streptacitin Beeds(购自常州天地人和生物科技有限公司，货号SA092005)进行StrepTactin纯化，并用Biotin洗脱后，利用SDS-PAGE凝胶电泳进行分离，结果显示：在还原状态，表达的融合蛋白是单体形式；在非还原状态，表达的融合蛋白是二聚体结构。In some embodiments, 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.
1.3靶向CD3促进CD3内吞的融合蛋白1.3 Fusion protein targeting CD3 to promote CD3 endocytosis
在一些实施方式中，本实施例设计了靶向CD3促进CD3内吞的融合蛋白BMC-STII-VXT，其包括靶向CD3的单链抗体片段BMC(scFv)，Strep tag II标签，甘丝氨酸铰链区(Glycine-Serine linker)和来自胶原蛋白XV的三聚体结构域(VXT)，如图1C所示。In some embodiments, 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.
其中靶向CD3的单链抗体为特异性结合CD3受体蛋白的抗体BMC：Among them, the single-chain antibody targeting CD3 is the antibody BMC that specifically binds to the CD3 receptor protein:
该靶向CD3的单链抗体BMC的重链可变区的氨基酸序列如SEQ ID NO.15所示：
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:
其中，STII(Trp-Ser-His-Pro-Gln-Phe-Glu-Lys)是8个氨基酸的Strep Tag II标签，Strep-tag II对Strep-Tactin的结合亲和力比对streptavidin高将近100倍，因此可以用Strep-Tactin进行纯化；VXT是促进三聚体形成的组件，其来自人源XV型胶原蛋白的三聚体结构域(VXT的氨基酸序列如SEQ ID NO.18所示)。VXT结构域包含4个β-折叠结构和α-螺旋，可以形成三螺旋的结构。Among them, STII (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) is an 8-amino-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 β-sheet structures and α-helices, which can form a triple-helix structure.
1.4靶向CD5促进CD5内吞的融合蛋白1.4 Fusion protein targeting CD5 to promote CD5 endocytosis
在一些实施方式中，本实施例设计了靶向CD5促进CD5内吞的融合蛋白BMD-STII-VXT，其包括靶向CD5的单链抗体片段BMD(scFv)，Strep tag II标签，甘丝氨酸铰链区(Glycine-Serine linker)和来自胶原蛋白XV的三聚体结构域(VXT)，如图1D所示。In some embodiments, 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.
其中靶向CD5的单链抗体为特异性结合CD5受体蛋白的抗体BMD：The single-chain antibody targeting CD5 is an antibody BMD that specifically binds to the CD5 receptor protein:
该靶向CD5的单链抗体BMD的重链可变区的氨基酸序列如SEQ ID NO.221所示：

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:

其中，STII(Trp-Ser-His-Pro-Gln-Phe-Glu-Lys)是8个氨基酸的Strep Tag II标签，Strep-tag II对Strep-Tactin的结合亲和力比对streptavidin高将近100倍，因此可以用Strep-Tactin进行纯化；VXT是促进三聚体形成的组件，其来自人源XV型胶原蛋白的三聚体结构域(VXT的氨基酸序列如SEQ ID NO.18所示)。VXT结构域包含4个β-折叠结构和α-螺旋，可以形成三螺旋的结构。Among them, STII (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) is an 8-amino-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 β-sheet structures and α-helices, which can form a triple-helix structure.
1.5靶向CD7促进CD7内吞的融合蛋白1.5 Fusion protein targeting CD7 to promote CD7 endocytosis
在一些实施方式中，本实施例设计了靶向CD7促进CD7内吞的融合蛋白BME-STII-VXT，其包括靶向CD7的单链抗体片段BME(scFv)，Strep tag II标签，甘丝氨酸铰链区(Glycine-Serine linker)和来自胶原蛋白XV的三聚体结构域(VXT)，如图1E所示。In some embodiments, 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.
其中靶向CD7的单链抗体为特异性结合CD7受体蛋白的抗体BME：The single-chain antibody targeting CD7 is the antibody BME that specifically binds to the CD7 receptor protein:
该靶向CD7的单链抗体BME的重链可变区的氨基酸序列如SEQ ID NO.229所示：
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:
其中，STII(Trp-Ser-His-Pro-Gln-Phe-Glu-Lys)是8个氨基酸的Strep Tag II标签，Strep-tag II对Strep-Tactin的结合亲和力比对streptavidin高将近100倍，因此可以用Strep-Tactin进行纯化；VXT是促进三聚体形成的组件，其来自人源XV型胶原蛋白的三聚体结构域(VXT的氨基酸序列如SEQ ID NO.18所示)。VXT结构域包含4个β-折叠结构和α-螺旋，可以形成三螺旋的结构。Among them, STII (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) is an 8-amino-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 β-sheet structures and α-helices, which can form a triple-helix structure.
1.6靶向CD4促进CD4内吞的融合蛋白1.6 Fusion protein targeting CD4 to promote CD4 endocytosis
在一些实施方式中，本实施例设计了靶向CD4促进CD4内吞的融合蛋白BMF-STII-VXT，其包括靶向CD4的单链抗体片段BMF(scFv)，Strep tag II标签，甘丝氨酸铰链区(Glycine-Serine linker)和来自胶原蛋白XV的三聚体结构域(VXT)，如图1F所示。In some embodiments, 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.
其中靶向CD4的单链抗体为特异性结合CD4受体蛋白的抗体BMF：The single-chain antibody targeting CD4 is the antibody BMF that specifically binds to the CD4 receptor protein:
该靶向CD4的单链抗体BMF的重链可变区的氨基酸序列如SEQ ID NO.237所示：

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:

其中，STII(Trp-Ser-His-Pro-Gln-Phe-Glu-Lys)是8个氨基酸的Strep Tag II标签，Strep-tag II对Strep-Tactin的结合亲和力比对streptavidin高将近100倍，因此可以用Strep-Tactin进行纯化；VXT是促进三聚体形成的组件，其来自人源XV型胶原蛋白的三聚体结构域(VXT的氨基酸序列如SEQ ID NO.18所示)。VXT结构域包含4个β-折叠结构和α-螺旋，可以形成三螺旋的结构。Among them, STII (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) is an 8-amino-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 β-sheet structures and α-helices, which can form a triple-helix structure.
1.7靶向CD20促进CD20内吞的融合蛋白1.7 Fusion protein targeting CD20 to promote CD20 endocytosis
在一些实施方式中，本实施例设计了靶向CD20促进CD20内吞的融合蛋白BMF-STII-VXT，其包括靶向CD20的单链抗体片段BMG(scFv)，Strep tag II标签，甘丝氨酸铰链区(Glycine-Serine linker)和来自胶原蛋白XV的三聚体结构域(VXT)，如图1G所示。In some embodiments, 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.
其中靶向CD20的单链抗体为特异性结合CD20受体蛋白的抗体BMG：The single-chain antibody targeting CD20 is the antibody BMG that specifically binds to the CD20 receptor protein:
该靶向CD20的单链抗体BMF的重链可变区的氨基酸序列如SEQ ID NO.245所示：
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:
其中，STII(Trp-Ser-His-Pro-Gln-Phe-Glu-Lys)是8个氨基酸的Strep Tag II标签，Strep-tag II对Strep-Tactin的结合亲和力比对streptavidin高将近100倍，因此可以用Strep-Tactin进行纯化；VXT是促进三聚体形成的组件，其来自人源XV型胶原蛋白的三聚体结构域(VXT的氨基酸序列如SEQ ID NO.18所示)。VXT结构域包含4个β-折叠结构和α-螺旋，可以形成三螺旋的结构。Among them, STII (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) is an 8-amino-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 β-sheet structures and α-helices, which can form a triple-helix structure.
1.8靶向CD22促进CD22内吞的融合蛋白1.8 Fusion protein targeting CD22 to promote CD22 endocytosis
在一些实施方式中，本实施例设计了靶向CD22促进CD22内吞的融合蛋白BMF-STII-VXT，其包括靶向CD22的单链抗体片段BMH(scFv)，Strep tag II标签，甘丝氨酸铰链区(Glycine-Serine linker)和来自胶原蛋白XV的三聚体结构域(VXT)，如图1H所示。In some embodiments, 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.
其中靶向CD22的单链抗体为特异性结合CD22受体蛋白的抗体BMH：The single-chain antibody targeting CD22 is the antibody BMH that specifically binds to the CD22 receptor protein:
该靶向CD22的单链抗体BMF的重链可变区的氨基酸序列如SEQ ID NO.253所示：
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:
轻链可变区的氨基酸序列如SEQ ID NO.254所示：
The amino acid sequence of the light chain variable region is shown in SEQ ID NO. 254:
其中，STII(Trp-Ser-His-Pro-Gln-Phe-Glu-Lys)是8个氨基酸的Strep Tag II标签，Strep-tag II对Strep-Tactin的结合亲和力比对streptavidin高将近100倍，因此可以用Strep-Tactin进行纯化；VXT是促进三聚体形成的组件，其来 自人源XV型胶原蛋白的三聚体结构域(VXT的氨基酸序列如SEQ ID NO.18所示)。VXT结构域包含4个β-折叠结构和α-螺旋，可以形成三螺旋的结构。Among them, STII (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) is an 8-amino-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 β-sheet structures and α-helices, which can form a triple-helix structure.
1.9靶向CD64促进CD64内吞的融合蛋白1.9 Fusion protein targeting CD64 to promote CD64 endocytosis
在一些实施方式中，本实施例设计了靶向CD64促进CD64内吞的融合蛋白BMF-STII-VXT，其包括靶向CD64的单链抗体片段BMI(scFv)，Strep tag II标签，甘丝氨酸铰链区(Glycine-Serine linker)和来自胶原蛋白XV的三聚体结构域(VXT)，如图1I所示。In some embodiments, 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.
其中靶向CD64的单链抗体为特异性结合CD64受体蛋白的抗体BMI：The single-chain antibody targeting CD64 is the antibody BMI that specifically binds to the CD64 receptor protein:
该靶向CD64的单链抗体BMF的重链可变区的氨基酸序列如SEQ ID NO.261所示：
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:
轻链可变区的氨基酸序列如SEQ ID NO.262所示：
The amino acid sequence of the light chain variable region is shown in SEQ ID NO. 262:
其中，STII(Trp-Ser-His-Pro-Gln-Phe-Glu-Lys)是8个氨基酸的Strep Tag II标签，Strep-tag II对Strep-Tactin的结合亲和力比对streptavidin高将近100倍，因此可以用Strep-Tactin进行纯化；VXT是促进三聚体形成的组件，其来自人源XV型胶原蛋白的三聚体结构域(VXT的氨基酸序列如SEQ ID NO.18所示)。VXT结构域包含4个β-折叠结构和α-螺旋，可以形成三螺旋的结构。Among them, STII (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) is an 8-amino-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 β-sheet structures and α-helices, which can form a triple-helix structure.
实施例2：靶向TCR的融合蛋白与T细胞特异性结合Example 2: TCR-targeting fusion protein specifically binds to T cells
本实施例研究实施例1制备的BMA-STII-VXT融合蛋白与T细胞特异性结合的能力。This example studies the ability of the BMA-STII-VXT fusion protein prepared in Example 1 to specifically bind to T cells.
2.1 T细胞制备2.1 T cell preparation
利用健康人捐献的全血来分离外周血单个核细胞。利用淋巴细胞密度梯度分离法分离单个核细胞。分离的单个核细胞由CD3和CD28的抗体激活后得到T细胞。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.
2.2 BMA-STII-VXT融合蛋白与T细胞结合能力的检测2.2 Detection of the binding ability of BMA-STII-VXT fusion protein to T cells
对实施例1制备的BMA-STII-VXT融合蛋白进行Biotin修饰，然后将其与T细胞孵育，之后对T细胞进行流式细胞检测，共进行了5组实验来检测BMA-STII-VXT融合蛋白与T细胞的结合能力，实验过程如下所示：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:
A.以没有任何染色的T细胞作为对照组(流式检测结果如图2A所示)。A. Use T cells without any staining as the control group (flow cytometry results are shown in Figure 2A).
B.0.5M T细胞先标记BMA-STII-VXT-Biotin 1μL 30分钟，然后洗脱，之后再用CD8-Pacific blue(ThermoFisher，货号:MHCD0828)，Streptavidin-PE(Biolegend，货号:405204)染色15分钟，洗脱后进行流式检测(流式检测结果如图2B所示)；B.0.5M T cells are first labeled with 1μL 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);
C.0.5M T细胞先标记BMA-STII-VXT-Biotin 3μL 30分钟，然后洗脱，之后再用CD8-Pacific blue(ThermoFisher，货号:MHCD0828)，Streptavidin-PE(Biolegend，货号:405204)染色15分钟，洗脱后进行流式检测(流式检测结果如图2C所示)；C.0.5M T cells were first labeled with 3μL 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);
D.0.5M T细胞先标记BMA-STII-VXT-Biotin 3μL 30分钟，然后洗脱后，再用CD8-Pacific blue(ThermoFisher，货号:MHCD0828)，TCR-αβ-APC-Cy7(biolegend，货号:109220)染色15分钟，洗脱后进行流式检测(流式检测结果如图2D所示)；D.0.5M T cells were first labeled with 3μL of BMA-STII-VXT-Biotin for 30 minutes, then eluted, and then used CD8-Pacific blue (ThermoFisher, Catalog No.: MHCD0828), TCR-αβ-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细胞先标记BMA-STII-VXT-Biotin 3μL 30分钟，然后洗脱后，再用CD8-Pacific blue(ThermoFisher，货号:MHCD0828)，Streptavidin-PE(Biolegend，货号:405204),TCR-αβ-APC-Cy7(biolegend，货号:109220)染色15分钟，洗脱后进行流式检测(流式检测结果如图2E所示)。E.0.5M T cells are first labeled with 3μL 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 -αβ-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).
上述结果显示BMA-VXT能够高效结合到T细胞表面，并能与TCR-αβ的抗体竞争性结合T细胞表面的TCR.The above results show that BMA-VXT can efficiently bind to the surface of T cells and competitively bind to TCR on the surface of T cells with antibodies against TCR-αβ.
实施例3：靶向TCR的融合蛋白促进TCR内吞Example 3: Fusion protein targeting TCR promotes TCR endocytosis
本实施例研究实施例1制备的BMA-STII-VXT融合蛋白促进TCR内吞的能力。This example studies the ability of the BMA-STII-VXT fusion protein prepared in Example 1 to promote TCR endocytosis.
对实施例1制备的BMA-STII-VXT融合蛋白进行Biotin修饰后，和TCR-αβ抗体同时标记T细胞后进行孵育，之后进行流式细胞检测。T细胞的制备方法如实施例2中所示。共进行了四组实验，实验过程如下所示： After the BMA-STII-VXT fusion protein prepared in Example 1 was modified with Biotin, T cells were simultaneously labeled with TCR-αβ 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:
A.利用TCR-αβ-APC-Cy7(biolegend，货号:109220),标记对照组T细胞15分钟，清洗离心后进行流式检测；结果显示95.5％的细胞都呈现TCR-ab高表达(流式检测结果如图3A所示)；A. Use TCR-αβ-APC-Cy7 (biolegend, Cat. No.: 109220) to label T cells in the control group for 15 minutes, wash and centrifuge for flow cytometric detection; the results show that 95.5% of the cells show high expression of TCR-ab (flow cytometry The test results are shown in Figure 3A);
B.利用BMA-STII-VXT-Biotin标记T细胞30分钟，洗脱后孵育30分钟，再用Streptavidin-PE(biolegend，货号:405204)和TCR-αβ-APC-Cy7(biolegend，货号:109220),染色15分钟，洗脱后再进行流式检测；结果显示BMA-STII-VXT能完全屏蔽TCR-αβ抗体，竞争结合T细胞(流式检测结果如图3B所示)；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-αβ-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-αβ antibodies and compete for binding to T cells (flow cytometry results are shown in Figure 3B);
C.利用BMA-STII-VXT-Biotin标记T细胞30分钟，洗脱后孵育3小时，再染Streptavidin-PE(biolegend，货号:405204)和TCR-ab-APC-Cy7(biolegend，货号:109220)染色15分钟,洗脱后进行流式检测；结果显示TCR-αβ抗体不能结合T细胞，同时，BMA-STII-VXT的信号也消失，说明结合TCR的BMA-STII-VXT被内吞到T细胞内(流式检测结果如图3C所示)；C. Use BMA-STII-VXT-Biotin to label T cells for 30 minutes, incubate for 3 hours after elution, and then stain with Streptavidin-PE (biolegend, Cat. No.: 405204) and TCR-ab-APC-Cy7 (biolegend, Cat. No.: 109220) Stain for 15 minutes and perform flow cytometry after elution; the results show that the TCR-αβ antibody cannot bind to T cells. At the same time, the signal of BMA-STII-VXT also disappears, indicating that the BMA-STII-VXT that binds to TCR is endocytosed into T cells. Within (flow cytometry results are shown in Figure 3C);
D.T细胞进行BMA-STII-VXT-Biotin标记30分钟，洗脱后孵育6小时，再染Streptavidin-PE(biolegend，货号:405204和TCR-αβ-APC-Cy7(biolegend，货号:109220)染色15分钟,洗脱后进行流式检测；结果显示BMA-STII-VXT结合的信号也消失，但TCR-αβ抗体又重新结合到T细胞表面，说明结合TCR的BMA-STII-VXT被内吞到T细胞内，部分TCR经过6个小时又重新回到T细胞表面(流式检测结果如图3D所示)。D. 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-αβ-APC-Cy7 (biolegend, Cat. No.: 109220) for 15 minutes. , flow cytometry was performed after elution; the results showed that the BMA-STII-VXT binding signal also disappeared, but the TCR-αβ antibody rebinded to the T cell surface, indicating that the TCR-bound BMA-STII-VXT was endocytosed into the T cell. Within 6 hours, some TCRs returned to the T cell surface (flow cytometry results are shown in Figure 3D).
上述结果显示BMA-STII-VXT能完全屏蔽TCR-αβ抗体，使TCR-αβ抗体不能结合T细胞，同时也能促进BMA-STII-VXT结合的TCR被内吞到T细胞内，而未与BMA-STII-VXT结合的TCR在经过一段时间后又重新回到T细胞表面，从而可以与抗TCRαβ的抗体结合。The above results show that BMA-STII-VXT can completely shield TCR-αβ antibodies so that TCR-αβ 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-TCRαβ antibodies.
实施例4：利用靶向TCR的融合蛋白与LNP偶联递送药物Example 4: Drug delivery using TCR-targeting fusion protein conjugated with LNP
4.1靶向投递具有生物活性的核酸4.1 Targeted delivery of biologically active nucleic acids
通过将LNP与靶向TCR的融合蛋白直接或通过接头间接连接(结构如图4所示)，靶向投递具有生物活性的核酸进入T细胞，By connecting LNP to a TCR-targeting fusion protein directly or indirectly through a linker (the structure is shown in Figure 4), biologically active nucleic acids are targeted and delivered into T cells.
4.1.1靶向投递CAR4.1.1 Targeted delivery of CAR
通过将LNP与靶向TCR的融合蛋白直接或通过接头间接连接，所述LNP中包含表达特定功能蛋白从而改造T细胞成为CAR-T细胞的核酸，从而实现对肿瘤或病毒感染类疾病的治疗效果。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. .
4.1.2靶向投递CNK复合体4.1.2 Targeted delivery of CNK complex
通过将LNP与靶向TCR的融合蛋白直接或通过接头间接连接，所述LNP中包含表达特定功能蛋白从而改造T细胞成为CNK-UT细胞的核酸，从而实现对肿瘤或病毒感染类疾病的治疗效果。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. .
4.1.3靶向投递嵌合抗原构建体4.1.3 Targeted delivery of chimeric antigen constructs
通过将LNP与靶向TCR的融合蛋白直接或通过接头间接连接，所述LNP中包含表达特定功能蛋白从而改造T细胞成为表达嵌合蛋白构建体TPD的核酸，从而实现对肿瘤或病毒感染类疾病的治疗效果。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.
4.1.4靶向投递转座子4.1.4 Targeted delivery of transposons
通过将LNP与靶向TCR的融合蛋白直接或通过接头间接连接，所述LNP中包含所述具有生物活性的核酸为编码转座子的核酸，所述核酸包括在前述嵌合蛋白受体(CAR)、CNK-UT复合体或嵌合蛋白构建体两端嵌有反向重复序列的核酸和编码转座酶的核酸，使T细胞表达特定蛋白质(过程如图5所示)，从而实现对肿瘤或病毒感染类疾病的治疗效果。By connecting the LNP to the TCR-targeting fusion protein directly or indirectly through a linker, 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). ), 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.
4.2靶向投递化疗药物4.2 Targeted delivery of chemotherapy drugs
通过将LNP与靶向TCR的融合蛋白直接或通过接头间接连接(结构如图6所示)，靶向投递化疗药物进入T细胞，从而实现对肿瘤或病毒感染类疾病的治疗效果。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.
实施例5：利用靶向TCR的融合蛋白制作ADCExample 5: Preparation of ADC using TCR-targeting fusion protein
通过将小分子药物例如微管蛋白聚合抑制剂或DNA损伤剂缀合靶向TCR的融合蛋白，从而将小分子药物靶向投递进入T细胞，直接达到杀伤T细胞的功能，可用于治疗白血病、成人晚期癌症、胰腺癌、不可切除性胰腺癌、结肠直肠癌、转移性结肠直肠癌、卵巢癌、三阴性乳腺癌、造血/淋巴癌、结肠癌肝转移、小细胞肺癌、非小细胞肺癌、B细胞淋巴瘤、复发性或难治性B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、弥漫性大细胞淋巴瘤、复发性或难治性弥漫性大细胞淋巴瘤、间变性大细胞淋巴瘤、原发性纵隔B细胞淋巴瘤、复发性纵隔大B细胞淋巴瘤、难治性纵隔大B细胞淋巴瘤、大B细胞淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、复发性或难治性非霍奇金淋巴瘤、难治性侵袭性非霍奇金淋巴瘤、B细胞非霍奇金淋巴瘤、难治性非霍奇金淋巴瘤、结肠直肠上皮癌、胃癌、胰腺癌、三阴性浸润性乳腺癌、肾细胞癌、肺鳞状细胞癌、肝细胞癌、尿路上皮癌、白血病、B细胞白血病、B细胞急性淋巴细胞白血病、B细胞急性成淋巴细胞白血病、成人急性成淋巴细胞白血病、B细胞幼淋巴细胞白血病、儿童急性成淋巴细胞白血病、难治性儿童急性成淋巴细胞白血病、急性白血病、急性成淋巴细胞白血病、急性淋巴细胞白血病、幼淋巴细胞白血病、慢性淋巴细胞白血病、急性髓性白血病、复发性浆细胞骨髓瘤、难治性浆细胞骨髓瘤、多发性骨髓瘤、复发性或难治性多发性骨髓瘤、骨多发性骨髓瘤、脑恶性神经胶质 瘤、骨髓增生异常综合征、EGFR阳性结肠直肠癌、多形性胶质母细胞瘤、赘生物、母细胞性浆细胞样树突状细胞肿瘤、肝转移、实体瘤、晚期实体瘤、间皮素阳性肿瘤、血液恶性肿瘤及其他晚期恶性肿瘤。By conjugating small molecule drugs such as tubulin polymerization inhibitors or DNA damaging agents to TCR-targeting fusion proteins, 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 mediastinal large B-cell lymphoma, large B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma , relapsed or refractory non-Hodgkin lymphoma, refractory aggressive non-Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, refractory non-Hodgkin lymphoma, colorectal epithelial cancer, Gastric cancer, pancreatic cancer, triple-negative invasive breast cancer, renal cell carcinoma, lung squamous cell carcinoma, hepatocellular carcinoma, urothelial carcinoma, leukemia, B-cell leukemia, B-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic Leukemia, adult acute lymphoblastic leukemia, B-cell prolymphoblastic leukemia, childhood acute lymphoblastic leukemia, refractory childhood acute lymphoblastic leukemia, acute leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, prolymphoblastic leukemia Leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, relapsed plasma cell myeloma, refractory plasma cell myeloma, multiple myeloma, relapsed or refractory multiple myeloma, bone multiple myeloma, brain malignant glia neoplasms, myelodysplastic syndromes, EGFR-positive colorectal cancer, glioblastoma multiforme, neoplasia, blastic plasmacytoid dendritic cell neoplasms, liver metastases, solid tumors, advanced solid tumors, mesothelial protein-positive tumors, hematological malignancies and other advanced malignant tumors.
实施例6：利用靶向TCR的融合蛋白标记T细胞Example 6: Labeling T cells using fusion proteins targeting TCR
在靶向TCR的融合蛋白上连接氧化铁纳米颗粒，实现对T细胞的标记，从而在体外可以实现对回输体内的T细胞的体内检测和监测，从而了解T细胞的迁徙、分布，评估T细胞的功能，比如靶向性和在持续存留性(persistence)。 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.

Claims (38)

  1. 一种融合蛋白，其包含与抗原结合的组件(module)和促进三聚体形成的组件，所述抗原包含能实现网格蛋白依赖性内吞机制(clathrin-dependent endocytosis，CDE)的细胞膜受体蛋白，A fusion protein comprising a module that binds to an antigen containing a cell membrane receptor that enables clathrin-dependent endocytosis (CDE) and a module that promotes trimer formation protein,
    优选的，所述细胞膜受体蛋白选自T细胞膜受体抗原、B细胞膜受体抗原、单核细胞等抗原呈递细胞(Antigen present cell)的膜受体抗原中的一种或多种；Preferably, the cell membrane receptor protein is selected from one or more of T cell membrane receptor antigens, B cell membrane receptor antigens, and membrane receptor antigens of antigen presenting cells (Antigen present cells) such as monocytes;
    优选的，所述T细胞膜受体抗原选自TCR、CD3、CD4、CD5、CD7、CCR5、CXCR4中的一种或多种；Preferably, the T cell membrane receptor antigen is selected from one or more of TCR, CD3, CD4, CD5, CD7, CCR5, and CXCR4;
    优选的，所述B细胞膜受体抗原选自CD20和CD22；Preferably, the B cell membrane receptor antigen is selected from CD20 and CD22;
    优选的，所述单核细胞等抗原呈递细胞的膜受体抗原选自Fc受体抗原；优选地，所述Fc受体抗原选自Fcα、FcγRI、FcγRII、FcγRIV、FcεRI和Fcα/μR中的一种或多种；优选地，Fcα为FcαRI，更优选地为CD89；优选地，FcγRI为CD64；优选地，FcγRII为FcγRIIB2(CD32)；优选地，FcεRI为CD89。Preferably, the membrane receptor antigen of the antigen-presenting cells such as monocytes is selected from the group consisting of Fc receptor antigens; preferably, the Fc receptor antigen is selected from the group consisting of Fcα, FcγRI, FcγRII, FcγRIV, FcεRI and Fcα/μR. One or more; preferably, Fcα is FcαRI, more preferably CD89; preferably, FcγRI is CD64; preferably, FcγRII is FcγRIIB2 (CD32); preferably, FcεRI is CD89.
  2. 根据权利要求1所述的融合蛋白，所述促进三聚体形成的组件选自脂联素或其胶原样结构域、T4纤维蛋白三聚体结构域、人类胶原蛋白的三聚体结构，如：穿膜胶原蛋白XIII、XV、XVII和XVIII的氨基端NC1结构域、原纤维相关胶原蛋白IX、XII、XIV、XVI、XIX、XX和XXI的NC2结构域、GCN4三聚化结构域、人肺表面活性剂蛋白A(SP-A)、甘露糖结合蛋白A(MBP-A)、大肠杆菌天冬氨酸转氨甲酰酶(ATCase)的催化亚基、寡聚卷曲螺旋粘附素和包膜病毒I类融合蛋白的互补七肽重复区；The fusion protein according to claim 1, the 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 : N-terminal NC1 domain of transmembrane collagens XIII, XV, XVII and XVIII, NC2 domain of fibril-associated collagens IX, XII, XIV, XVI, XIX, XX and XXI, GCN4 trimerization domain, human Pulmonary surfactant protein A (SP-A), mannose-binding protein A (MBP-A), the catalytic subunit of Escherichia coli aspartate transcarbamylase (ATCase), oligomeric coiled-coil adhesins, and Complementary heptapeptide repeat region of enveloped virus class I fusion protein;
    优选的，所述促进三聚体形成的组件包含XV的氨基端NC1结构域；Preferably, the component that promotes trimer formation includes the amino-terminal NC1 domain of XV;
    优选地，所述促进三聚体形成的组件包含与SEQ ID NO:18所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；优选地，其氨基酸序列为如SEQ ID NO.18所示的氨基酸序列或者在SEQ ID NO:18上缺失、添加或取代1、2或3个残基所得到的氨基酸序列。Preferably, 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%, 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, its amino acid sequence is the amino acid sequence shown in SEQ ID NO. 18 or in SEQ ID The amino acid sequence obtained by deleting, adding or substituting 1, 2 or 3 residues on NO:18.
  3. 根据权利要求1或2所述的融合蛋白，其中与抗原结合的组件选自抗原的抗体或其抗原结合片段或与所述膜受体蛋白结合的配体。The fusion protein according to claim 1 or 2, wherein the 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.
  4. 根据权利要求1-3中任一项所述的融合蛋白，其中所述TCR抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.1或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.2或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.3或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO.4或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO.5或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.6或其任何变体的轻链CDR3。The fusion protein according to any one of claims 1-3, wherein the antibody of the TCR antigen or the 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 heavy chain CDR2 of the amino acid sequence SEQ ID NO.2 or any variant thereof is selected from the heavy chain CDR3 of the amino acid sequence SEQ ID NO.3 or any variant thereof; and/or is selected from the heavy chain CDR3 of the amino acid sequence SEQ ID NO.4 or any variant thereof; The light chain CDR1 of any variant is selected from the light chain CDR2 of the amino acid sequence SEQ ID NO. 5 or any variant thereof, and the light chain CDR3 is selected from the amino acid sequence SEQ ID NO. 6 or any variant thereof.
  5. 根据权利要求1-4中任一项所述的融合蛋白，其中所述TCR抗原的抗体或其抗原结合片段包含氨基酸序列如SEQ ID NO:1、2、3所示的重链CDR1、CDR2及CDR3，和/或分别包含氨基酸序列如SEQ ID NO:4、5、6所示的轻链CDR1、CDR2及CDR3。The fusion protein according to any one of claims 1-4, wherein the antibody of the TCR antigen or the antigen-binding fragment thereof includes the heavy chain CDR1, CDR2 and amino acid sequence shown in SEQ ID NO: 1, 2, and 3. CDR3, and/or light chain CDR1, CDR2 and CDR3 respectively comprising amino acid sequences as shown in SEQ ID NO: 4, 5 and 6.
  6. 根据权利要求1-5中任一项所述的融合蛋白，其中所述TCR抗体片段包含选自氨基酸序列SEQ ID NO.7或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO.8或其任何变体的轻链可变区。The fusion protein according to any one of claims 1-5, wherein the 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. The light chain variable region of sequence SEQ ID NO. 8 or any variant thereof.
  7. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD3抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.9或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.10或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.11或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO.12或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO.13或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.14或其任何变体的轻链CDR3。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD3 antigen or the antigen-binding fragment thereof comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO. 9 or any variant thereof, selected from The heavy chain CDR2 of the amino acid sequence SEQ ID NO.10 or any variant thereof is selected from the heavy chain CDR3 of the amino acid sequence SEQ ID NO.11 or any variant thereof; and/or is selected from the amino acid sequence SEQ ID NO.12 or the heavy chain CDR3 of any variant thereof; The light chain CDR1 of any variant is selected from the light chain CDR2 of the amino acid sequence SEQ ID NO. 13 or any variant thereof, and the light chain CDR3 is selected from the amino acid sequence SEQ ID NO. 14 or any variant thereof.
  8. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD3抗原的抗体或其抗原结合片段包含氨基酸序列如SEQ ID NO:9、10、11所示的重链CDR1、CDR2及CDR3，和/或分别包含氨基酸序列如SEQ ID NO:12、13、14所示的轻链CDR1、CDR2及CDR3。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD3 antigen or the antigen-binding fragment thereof includes the heavy chain CDR1, CDR2 and amino acid sequence shown in SEQ ID NO: 9, 10, 11 and CDR3, and/or light chain CDR1, CDR2 and CDR3 respectively comprising the amino acid sequences shown in SEQ ID NO: 12, 13 and 14.
  9. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD3抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.15或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO.16或其任何变体的轻链可变区。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD3 antigen or the 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 a light chain variable region selected from the amino acid sequence SEQ ID NO. 16 or any variant thereof.
  10. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD5抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.215或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.216或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.217或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO.218或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO.219或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.220或其任何变体的轻链CDR3。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD5 antigen or the antigen-binding fragment thereof comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO. 215 or any variant thereof, selected from The heavy chain CDR2 of the amino acid sequence SEQ ID NO.216 or 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 is selected from the heavy chain CDR3 of the amino acid sequence SEQ ID NO.218 or any variant thereof; The light chain CDR1 of any variant is selected from the light chain CDR2 of the amino acid sequence SEQ ID NO. 219 or any variant thereof, and the light chain CDR3 is selected from the amino acid sequence SEQ ID NO. 220 or any variant thereof.
  11. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD5抗原的抗体或其抗原结合片段包含氨基酸序列如SEQ ID NO:215、216、217所示的重链CDR1、CDR2及CDR3，和/或分别包含氨基酸序列如SEQ ID NO:218、219、220所示的轻链CDR1、CDR2及CDR3。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD5 antigen or the antigen-binding fragment thereof includes the heavy chain CDR1, CDR2 and amino acid sequence shown in SEQ ID NO: 215, 216, 217 and CDR3, and/or light chain CDR1, CDR2 and CDR3 respectively comprising the amino acid sequences shown in SEQ ID NO: 218, 219 and 220.
  12. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD5抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.221或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO.222或其任何变体的轻链可 变区。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD5 antigen or the antigen-binding fragment thereof comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO. 221 or any variant thereof, and/or the light chain selected from the amino acid sequence SEQ ID NO. 222 or any variant thereof may Change area.
  13. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD7抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.223或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.224或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.225或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO.226或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO.227或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.228或其任何变体的轻链CDR3。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD7 antigen or the antigen-binding fragment thereof comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO. 223 or any variant thereof, selected from The heavy chain CDR2 of the amino acid sequence SEQ ID NO.224 or 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 is selected from the amino acid sequence SEQ ID NO.226 or the heavy chain CDR3 of any variant thereof; The light chain CDR1 of any variant is selected from the light chain CDR2 of the amino acid sequence SEQ ID NO. 227 or any variant thereof, and the light chain CDR3 is selected from the amino acid sequence SEQ ID NO. 228 or any variant thereof.
  14. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD7抗原的抗体或其抗原结合片段包含氨基酸序列如SEQ ID NO:223、224、225所示的重链CDR1、CDR2及CDR3，和/或分别包含氨基酸序列如SEQ ID NO:226、227、228所示的轻链CDR1、CDR2及CDR3。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD7 antigen or the antigen-binding fragment thereof comprises the heavy chain CDR1, CDR2 and amino acid sequence shown in SEQ ID NO: 223, 224, 225 and CDR3, and/or light chain CDR1, CDR2 and CDR3 respectively comprising the amino acid sequences shown in SEQ ID NO: 226, 227 and 228.
  15. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD7抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.229或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO.230或其任何变体的轻链可变区。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD7 antigen or the antigen-binding fragment thereof comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO. 229 or any variant thereof, and/or a light chain variable region selected from the amino acid sequence SEQ ID NO. 230 or any variant thereof.
  16. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD4抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.231或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.232或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.233或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO.234或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO.235或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.235或其任何变体的轻链CDR3。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD4 antigen or the antigen-binding fragment thereof comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO. 231 or any variant thereof, selected from The heavy chain CDR2 of the amino acid sequence SEQ ID NO.232 or 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 is selected from the amino acid sequence SEQ ID NO.234 or the heavy chain CDR3 of any variant thereof; The light chain CDR1 of any variant is selected from the light chain CDR2 of the amino acid sequence SEQ ID NO. 235 or any variant thereof, and the light chain CDR3 is selected from the amino acid sequence SEQ ID NO. 235 or any variant thereof.
  17. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD4抗原的抗体或其抗原结合片段分别包含氨基酸序列如SEQ ID NO:231、232、233所示的重链CDR1、CDR2及CDR3，和/或分别包含氨基酸序列如SEQ ID NO:234、235、236所示的轻链CDR1、CDR2及CDR3。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD4 antigen or the antigen-binding fragment thereof respectively includes the heavy chain CDR1 and CDR2 of the amino acid sequence shown in SEQ ID NO: 231, 232, and 233. and CDR3, and/or light chain CDR1, CDR2 and CDR3 respectively comprising amino acid sequences as shown in SEQ ID NO: 234, 235 and 236.
  18. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD4抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.237或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO.238或其任何变体的轻链可变区。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD4 antigen or the antigen-binding fragment thereof comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO. 237 or any variant thereof, and/or a light chain variable region selected from the amino acid sequence SEQ ID NO. 238 or any variant thereof.
  19. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD20抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.239或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.240或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.241或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO.242或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO.243或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.244或其任何变体的轻链CDR3。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD20 antigen or the antigen-binding fragment thereof comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO. 239 or any variant thereof, selected from The heavy chain CDR2 of the amino acid sequence SEQ ID NO.240 or 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 is selected from the heavy chain CDR3 of the amino acid sequence SEQ ID NO.242 or any variant thereof; The light chain CDR1 of any variant is selected from the light chain CDR2 of the amino acid sequence SEQ ID NO. 243 or any variant thereof, and the light chain CDR3 is selected from the amino acid sequence SEQ ID NO. 244 or any variant thereof.
  20. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD20抗原的抗体或其抗原结合片段分别包含氨基酸序列如SEQ ID NO:239、240、241所示的重链CDR1、CDR2及CDR3，和/或分别包含氨基酸序列如SEQ ID NO:242、243、244所示的轻链CDR1、CDR2及CDR3。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD20 antigen or the antigen-binding fragment thereof respectively includes the heavy chain CDR1 and CDR2 of the amino acid sequence shown in SEQ ID NO: 239, 240, and 241. and CDR3, and/or light chain CDR1, CDR2 and CDR3 respectively comprising amino acid sequences as shown in SEQ ID NO: 242, 243 and 244.
  21. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD20抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.245或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO.246或其任何变体的轻链可变区。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD20 antigen or the antigen-binding fragment thereof comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO. 245 or any variant thereof, and/or a light chain variable region selected from the amino acid sequence SEQ ID NO. 246 or any variant thereof.
  22. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD22抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.247或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.248或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.249或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO.250或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO.251或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.252或其任何变体的轻链CDR3。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD22 antigen or the antigen-binding fragment thereof comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO. 247 or any variant thereof, selected from The heavy chain CDR2 of the amino acid sequence SEQ ID NO.248 or 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 is selected from the amino acid sequence SEQ ID NO.250 or the heavy chain CDR3 of any variant thereof; The light chain CDR1 of any variant is selected from the light chain CDR2 of the amino acid sequence SEQ ID NO. 251 or any variant thereof, and the light chain CDR3 is selected from the amino acid sequence SEQ ID NO. 252 or any variant thereof.
  23. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD22抗原的抗体或其抗原结合片段分别包含氨基酸序列如SEQ ID NO:247、248、249所示的重链CDR1、CDR2及CDR3，和/或分别包含氨基酸序列如SEQ ID NO:250、251、252所示的轻链CDR1、CDR2及CDR3。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD22 antigen or the antigen-binding fragment thereof respectively includes the heavy chain CDR1 and CDR2 of the amino acid sequence shown in SEQ ID NO: 247, 248, and 249. and CDR3, and/or light chain CDR1, CDR2 and CDR3 respectively comprising amino acid sequences as shown in SEQ ID NO: 250, 251 and 252.
  24. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD22抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.253或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO.254或其任何变体的轻链可变区。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD22 antigen or the antigen-binding fragment thereof comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO. 253 or any variant thereof, and/or a light chain variable region selected from the amino acid sequence SEQ ID NO. 254 or any variant thereof.
  25. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD64抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.255或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO.256或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO.257或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO.258或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO.259或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO.260或其任何变体的轻链CDR3。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD64 antigen or the antigen-binding fragment thereof comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO. 255 or any variant thereof, selected from The heavy chain CDR2 of the amino acid sequence SEQ ID NO.256 or 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 is selected from the amino acid sequence SEQ ID NO.258 or the heavy chain CDR3 of any variant thereof; The light chain CDR1 of any variant is selected from the light chain CDR2 of the amino acid sequence SEQ ID NO. 259 or any variant thereof, and the light chain CDR3 is selected from the amino acid sequence SEQ ID NO. 260 or any variant thereof.
  26. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD64抗原的抗体或其抗原结合片段分别包含氨基酸序列如SEQ ID NO:255、256、257所示的重链CDR1、CDR2及CDR3，和/或分别包含氨基酸序列如SEQ ID NO:258、259、260所示的轻链CDR1、CDR2及CDR3。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD64 antigen or the antigen-binding fragment thereof respectively includes the heavy chain CDR1 and CDR2 of the amino acid sequence shown in SEQ ID NO: 255, 256, and 257. and CDR3, and/or light chain CDR1, CDR2 and CDR3 respectively comprising the amino acid sequences shown in SEQ ID NO: 258, 259 and 260.
  27. 根据权利要求1-3中任一项所述的融合蛋白，其中所述CD64抗原的抗体或其抗原结合片段包含选自氨基酸序列SEQ ID NO.261或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO.262或其任何变体的轻链 可变区。The fusion protein according to any one of claims 1-3, wherein the antibody of the CD64 antigen or the antigen-binding fragment thereof comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO. 261 or any variant thereof, and/or a light chain selected from the amino acid sequence SEQ ID NO. 262 or any variant thereof Variable area.
  28. 根据权利要求1-27中任一项所述的融合蛋白，所述与抗原结合的组件和促进三聚体形成的组件可直接连接或通过接头L1连接；According to the fusion protein according to any one of claims 1-27, the component that binds to the antigen and the component that promotes trimer formation can be directly connected or connected through a linker L1;
    优选地，所述接头为柔性接头；Preferably, the joint is a flexible joint;
    优选地，所述融合蛋白还包含纯化标签；所述纯化标签在所述融合蛋白的N端、C端或在所述与抗原结合的组件和促进三聚体形成的组件之间；更优选地，所述纯化标签在与抗原结合的组件和促进三聚体形成的组件之间；Preferably, 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; more preferably , the purification tag is between a component that binds to the antigen and a component that promotes trimer formation;
    优选地，所述纯化标签选自GST标签、His标签、Myc标签、E标签、Strep标签和HA标签；更优选地所述Strep标签为STII标签，其氨基酸序列如SEQ ID NO.17所示；Preferably, the purification tag is selected from the group consisting of GST tag, His tag, Myc tag, E tag, Strep tag and HA tag; more preferably, the Strep tag is an STII tag, and its amino acid sequence is as shown in SEQ ID NO. 17;
    优选地，所述柔性接头L1选自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)、GGGGSGGGGSGGGGS(SEQ ID NO.24)、GGGGSGGGGSGGGG(SEQ ID NO.25)、GGGGSGGGGSGGGGSGGGGS(SEQ ID NO.26)、GGGGSGGGGSGGGSGGGGS(SEQ ID NO.27)、GSGSGSGS(SEQ ID NO.28)、GGSGSGSG(SEQ ID NO.29)、GGSGSG(SEQ ID NO.30)、GGSG(SEQ ID NO.31)；Preferably, 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), GGGGSGGGSGGGGGS(SEQ ID NO.24), GGGGSGGGGSGGGG(SEQ ID NO.25), GGGGSGGGGSGGGGSGGGGS(SEQ ID NO.26), GGGGSGGGGSGGGSGGGGS(SEQ ID NO.27), GSGSGSGS(SEQ ID NO.28), GGSGSGSG(SEQ ID NO.29), GGSGSG(SEQ ID NO.30), GGSG(SEQ ID NO.31);
    更优选地，所述接头L1选自GGGS(SEQ ID NO.19)、GGGGSGGGGS(SEQ ID NO.21)、GGGGSGGGGSGGGGSGGGGS(SEQ ID NO.26)和GGGGSGGGGSGGGSGGGGS(SEQ ID NO.27)。More preferably, 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).
  29. 根据权利要求1-6中任一项所述的融合蛋白，所述融合蛋白包含与SEQ ID NO:36所示氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列；优选地，所述氨基酸序列为如SEQ ID NO:36所示的氨基酸序列或者在SEQ ID NO:36上缺失、添加或取代1、2或3个残基所得到的氨基酸序列。The fusion protein according to any one of claims 1-6, said fusion protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 36, 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%; Preferably, the amino acid sequence is such as SEQ ID NO: 36 The amino acid sequence shown or the amino acid sequence obtained by deleting, adding or substituting 1, 2 or 3 residues on SEQ ID NO:36.
  30. 编码权利要求1-29中任一项所述的融合蛋白的核酸分子。Nucleic acid molecule encoding the fusion protein of any one of claims 1-29.
  31. 包含权利要求30所述核酸分子的载体。A vector comprising the nucleic acid molecule of claim 30.
  32. 包含权利要求30所述的核酸分子或权利要求31所述载体的细胞。A cell comprising the nucleic acid molecule of claim 30 or the vector of claim 31.
  33. 一种组合物，其包含权利要求1-29中任一项所述的融合蛋白、权利要求30所述的核酸、权利要求31所述的载体和/或权利要求32所述的细胞。A composition comprising the fusion protein of any one of claims 1-29, the nucleic acid of claim 30, the vector of claim 31, and/or the cell of claim 32.
  34. 一种前药，其包含选自下述任一：A prodrug comprising any one selected from:
    (1)权利要求1-26中任一项所述的融合蛋白和LNP；(1) The fusion protein and LNP according to any one of claims 1-26;
    优选地，其中所述融合蛋白和所述LNP通过接头L2连接；更优选地，所述接头L2为在柔性多肽的C末端加入了一个半胱氨酸残基的多肽；所述柔性多肽选自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)、GGGGSGGGGSGGGGS(SEQ ID NO.24)、GGGGSGGGGSGGGG(SEQ ID NO.25)、GGGGSGGGGSGGGGSGGGGS(SEQ ID NO.26)、GGGGSGGGGSGGGSGGGGS(SEQ ID NO.27)、GSGSGSGS(SEQ ID NO.28)、GGSGSGSG(SEQ ID NO.29)、GGSGSG(SEQ ID NO.30)、GGSG(SEQ ID NO.31)；Preferably, 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 flexible polypeptide 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),GGGGSGGGGSGGGGS(SEQ ID NO.23) .24),GGGGSGGGGSGGGG(SEQ ID NO.25),GGGGSGGGGSGGGGSGGGGS(SEQ ID NO.26),GGGGSGGGGSGGGSGGGGS(SEQ ID NO.27),GSGSGSGS(SEQ ID NO.28),GGSGSGSG(SEQ ID NO.29),GGSGSG (SEQ ID NO.30), GGSG(SEQ ID NO.31);
    更优选地，所述接头L2选自GGGSC(SEQ ID NO.32)、GGGGSGGGGSC(SEQ ID NO.33)、GGGGSGGGGSGGGGSGGGGSC(SEQ ID NO.34)和GGGGSGGGGSGGGSGGGGSC(SEQ ID NO.35)；More preferably, 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);
    优选地，所述LNP内包含至少一种具有生物活性的核酸、蛋白和小分子化合物；优选地，所述具有生物活性的核酸选自DNA和RNA中的一种或多种；优选地，所述小分子化合物为化疗药物；Preferably, 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 biologically active nucleic acid is selected from one or more of DNA and RNA. The small molecule compounds are chemotherapy drugs;
    更优选地，所述具有生物活性的核酸选自编码嵌合抗原受体(CAR)的核酸、编码嵌合自然杀伤细胞受体(CNK)复合体的核酸，编码促细胞凋亡蛋白的核酸和编码嵌合蛋白构建体的核酸中的一种或多种；More preferably, the biologically active nucleic acid is selected from the group consisting of a nucleic acid encoding a chimeric antigen receptor (CAR), a nucleic acid encoding a chimeric natural killer cell receptor (CNK) complex, a nucleic acid encoding a pro-apoptotic protein, and One or more nucleic acids encoding a chimeric protein construct;
    更优选地，所述具有生物活性的蛋白选自嵌合抗原受体(CAR)、嵌合自然杀伤细胞受体(CNK)复合体，促细胞凋亡蛋白和嵌合蛋白构建体中的一种或多种；More preferably, the biologically active protein is selected from one of chimeric antigen receptor (CAR), chimeric natural killer cell receptor (CNK) complex, pro-apoptotic protein and chimeric protein construct. or more;
    更优选地，所述小分子化合物选自化疗药物中的一种或多种；More preferably, the small molecule compound is selected from one or more chemotherapeutic drugs;
    优选地，其中，所述促细胞凋亡蛋白为Bcl-2家族蛋白，包括但不限于Bax、Bak、Bok、Bad、Bid、Bik、Bim、HrkBnip3、Nix/Bnip3L、Noxa和Puma；Preferably, 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;
    优选地，所述具有生物活性的核酸为编码转座子的核酸，所述核酸包括目的基因的核酸和编码转座酶的核酸；更优选地，所述目的基因选自前述嵌合蛋白受体(CAR)、嵌合自然杀伤细胞受体(CNK)复合体或嵌合蛋白构建体；Preferably, the biologically active nucleic acid is a nucleic acid encoding a transposon, and the nucleic acid includes a nucleic acid of a target gene and a nucleic acid encoding a transposase; more preferably, the target gene is selected from the aforementioned chimeric protein receptors. (CAR), chimeric natural killer cell receptor (CNK) complex or chimeric protein construct;
    优选地，所述具有生物活性的核酸是表达载体；Preferably, the biologically active nucleic acid is an expression vector;
    优选地，所述LNP内包含的化疗药物选自：细胞毒素、烷化剂、鬼臼类、喜树碱类、紫杉类、抗代谢类、抗生素类抗肿瘤药物中的一种或多种，可以列举的实例包括但不限于咪唑四嗪酮类药物，例如替莫唑胺；铂类药物，例如奥沙利铂、顺铂、卡铂、奈达铂、双环铂、乐铂、四硝酸三铂、菲铂、吡铂、沙铂；亚硝脲类，例如卡氮芥、环已亚硝脲、甲环亚硝脲、嘧啶亚硝脲、卡莫司汀、洛莫司汀、福莫司汀、尼莫司汀、雷莫司汀、链脲佐菌素；喜树碱类，例如喜树碱、羟基喜树碱、伊立替康、拓扑替康；长春碱类，例如长春瑞滨、长春碱、长春新碱、长春地辛、长春富宁；丙卡巴肼；盐酸米托蒽醌；氮芥类，例如氮芥、氮甲、环磷酰胺、异环磷酰胺、雌二醇氮芥、曲磷胺、苯丙氨酸氮芥、苯丁酸氮芥、美法仑、泼尼莫司汀、苯达莫司汀、乌拉莫司汀、雌莫司汀、噻 替派；烷基磺酸酯类，例如白消安、甘露舒凡、苏消安；氟嘧啶衍生物，例如吉西他滨、卡培他滨、氟尿嘧啶、双呋氟尿嘧啶、去氧氟尿苷、替加氟、卡莫氟、三氟尿苷；紫杉烷类，例如紫杉醇、白蛋白结合的紫杉醇、紫杉醇脂质体以及多西他赛；培美曲塞、依托泊苷、伊立替康、丝裂霉素、阿糖胞苷、阿扎胞苷、氨柔比星、甲氨蝶呤、表阿霉素、阿霉素、Sapacitabine、普那布林、曲奥舒凡、地匹福林盐酸盐、替吉奥和encequidar中的一种或两种或三种；Preferably, the chemotherapeutic drugs contained in the LNP are selected from: one or more of cytotoxins, alkylating agents, podophyllotoxins, camptothecins, taxanes, anti-metabolites, and antibiotic anti-tumor drugs. , examples that can be cited include, but are not limited to, imidazole tetrazinone drugs, such as temozolomide; platinum drugs, such as oxaliplatin, cisplatin, carboplatin, nedaplatin, bicycloplatin, leplatin, triplatinum tetranitrate, Phernanplatin, picoplatin, satraplatin; nitrosoureas, such as carmustine, cyclohexanenitrosourea, mesocycline nitrosourea, pyrimidine nitrosourea, carmustine, lomustine, formostine , Nimustine, Ramustine, Streptozotocin; Camptothecins, such as camptothecin, hydroxycamptothecin, irinotecan, topotecan; Vinblastines, such as vinorelbine, vinorelbine, Alkali, vincristine, vindesine, vinca funin; procarbazine; mitoxantrone hydrochloride; nitrogen mustards, such as nitrogen mustard, nitrogen mustard, cyclophosphamide, ifosfamide, estradiol nitrogen mustard, Trofosfamide, chlorambucil, chlorambucil, melphalan, prednimustine, bendamustine, uramustine, estramustine, thiamine Tepa; alkyl sulfonate esters, such as busulfan, mannosulfan, sulfan; fluoropyrimidine derivatives, such as gemcitabine, capecitabine, fluorouracil, furfururacil, deoxyfluridine, tiga Fluoride, carmofur, trifluridine; taxanes such as paclitaxel, albumin-bound paclitaxel, liposomal paclitaxel, and docetaxel; pemetrexed, etoposide, irinotecan, mitogen Amrubicin, cytarabine, azacitidine, amrubicin, methotrexate, epirubicin, doxorubicin, sapacitabine, plinabulin, trioxofan, dipifrine hydrochloride One or two or three of salt, tegio and encequidar;
    优选地，所述LNP内包含的细胞毒素选自海兔毒素(dolastatin)及奥瑞他汀(auristatin)类细胞毒分子，美登素(maytansine)类细胞毒分子；所述DNA损伤剂包括但不限于卡奇霉素类(calicheamicin)、倍癌霉素(duocarmycin)类、安曲霉素类衍生物PBD、喜树碱类衍生物SN-38；Preferably, the cytotoxin contained in the LNP is selected from the group consisting of dolastatin, auristatin-like cytotoxic molecules, and maytansine-like cytotoxic molecules; the DNA damaging agent includes but does not Limited to calicheamicin, duocarmycin, antromycin derivative PBD, and camptothecin derivative SN-38;
    更优选地，所述LNP内包含的细胞毒素选自瓢菌素(amanitins)、蒽环类物(anthracyclines)、浆果赤霉素(baccatins)、喜树碱(camptothecins)、西马多丁(cemadotins)、秋水仙碱(colchicines)、秋水仙胺(colcimids)、考布他汀(combretastatins)、隐菲辛(cryptophycins)、圆皮海绵内酯(discodermolides)、多烯紫杉醇(docetaxel)、阿霉素(doxorubicin)、棘霉素(echinomycins)、艾榴塞洛素(eleutherobins)、埃博霉素(epothilones)、雌莫司汀(estramustines)、偏端霉素(lexitropsins)、美登素(maytansines)、氨甲蝶呤(methotrexate)、纺锤菌素(netropsins)、嘌呤霉素(puromycins)、根瘤菌素(rhizoxins)、紫杉烷(taxanes)、微管蛋白裂解素(tubulysins)、或长春花生物碱(vincaalkaloids)；More preferably, the cytotoxin contained in the LNP is selected from the group consisting of amanitins, anthracyclines, baccatins, camptothecins, cemadotins ), colchicines, colcimids, combretastatins, cryptophycins, discodermolides, docetaxel, doxorubicin ( doxorubicin), echinomycins, eleuterobins, epothilones, estramustines, lexitropsins, maytansines, Methotrexate, netropsins, puromycins, rhizoxins, taxanes, tubulysins, or vinca alkaloids (vincaalkaloids);
    (2)权利要求1-26中任一项所述的融合蛋白与细胞毒素，其中所述融合蛋白和所述细胞毒素通过接头单元连接；(2) The fusion protein and cytotoxin according to any one of claims 1 to 26, wherein the fusion protein and the cytotoxin are connected through a linker unit;
    优选地，所述与融合蛋白通过接头单元连接的细胞毒素选自海兔毒素(dolastatin)及奥瑞他汀(auristatin)类细胞毒分子，美登素(maytansine)类细胞毒分子；所述DNA损伤剂包括但不限于卡奇霉素类(calicheamicin)、倍癌霉素(duocarmycin)类、安曲霉素类衍生物PBD、喜树碱类衍生物SN-38；Preferably, the cytotoxin connected to the fusion protein through a linker unit is selected from the group consisting of dolastatin, auristatin-like cytotoxic molecules, and maytansine-like cytotoxic molecules; the DNA damage Agents include but are not limited to calicheamicin, duocarmycin, antromycin derivative PBD, and camptothecin derivative SN-38;
    优选地，所述与融合蛋白通过接头单元连接的细胞毒素选自瓢菌素(amanitins)、蒽环类物(anthracyclines)、浆果赤霉素(baccatins)、喜树碱(camptothecins)、西马多丁(cemadotins)、秋水仙碱(colchicines)、秋水仙胺(colcimids)、考布他汀(combretastatins)、隐菲辛(cryptophycins)、圆皮海绵内酯(discodermolides)、多烯紫杉醇(docetaxel)、阿霉素(doxorubicin)、棘霉素(echinomycins)、艾榴塞洛素(eleutherobins)、埃博霉素(epothilones)、雌莫司汀(estramustines)、偏端霉素(lexitropsins)、美登素(maytansines)、氨甲蝶呤(methotrexate)、纺锤菌素(netropsins)、嘌呤霉素(puromycins)、根瘤菌素(rhizoxins)、紫杉烷(taxanes)、微管蛋白裂解素(tubulysins)、或长春花生物碱(vincaalkaloids)；Preferably, the cytotoxin connected to the fusion protein through a linker unit is selected from the group consisting of amanitins, anthracyclines, baccatins, camptothecins, and simado. cemadotins, colchicines, colcimids, combretastatins, cryptophycins, discodermolides, docetaxel, acetaminophen Doxorubicin, echinomycins, eleuterobins, epothilones, estramustines, lexitropsins, maytansines maytansines, methotrexate, netropsins, puromycins, rhizoxins, taxanes, tubulysins, or vinifera Flower alkaloids (vincaalkaloids);
    更优选地，所述接头单元选自马来酰亚胺基-己酰基-缬氨酸-瓜氨酸-p-氨基苄氧基(Maleimido-Caproyl-Valine-Citrulline-p-Aminobenzyloxy,mc-vc-pAB)、马来酰亚胺基己酰基(Maleimidocaproyl，mc)、三甘氨酸肽接头(triglycyl peptide linker)、3-马来酰亚胺-丙酸(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-pyrrol-1-yl)ethoxy)ethoxy)propanoate)、Mal-Tri-EG-OSu(2,5-dioxopyrrolidin-1-yl 3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethoxypro panoate)、Mal-Tetra-EG-OSu(2,5-dioxopyrrolidin-1-yl 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16-tetraoxa-4-azan onadecan-19-oate)、Br-di-EG-OSu(2,5-dioxopyrrolidin-1-yl 3(2-(2-(2-bromoacetamido)ethoxy)ethoxy)propanoate)、Py-ds-prp-OSu(2-5-dioxopyrrolidin-1-yl 3-(pyridine-2-yldisulfanyl)propanoate)、Py-ds-Prp-OPEP(perfluorophenyl 3-(pyridine-2-yldisulfanyl)propanoate)、Py-ds-dmBut-OSu(2,5-dioxopyrrolidin-1-yl 4-methyl-4-(pyridine-2-yldisulfanyl)、Py-ds-dmBut-OPF(perfluorophenyl 4-methyl-4-(pyridine-2-yldisulfanyl)pentanoate)、SMCC(N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate)、MBS(3-maleimidobenzoic acid N-hydroxysuccinimide ester)、SATA(S-(N-succinimidyl)thioacetate)、SPDP((N-succinimidyl 3-(2-pyridyldithio)propionate)、SMPT((N-succinimidyloxy carbonyl)-1-methyl-1-(2-pyridyldithio)toluene)；More preferably, the linker unit is selected from Maleimido-Caproyl-Valine-Citrulline-p-Aminobenzyloxy (Maleimido-Caproyl-Valine-Citrulline-p-Aminobenzyloxy, mc-vc -pAB), maleimidocaproyl (mc), triglycyl peptide linker, 3-maleimido-propionic acid (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-pyrrol-1-yl)ethoxy)ethoxy)propanoate), Mal-Tri-EG-OSu(2,5-dioxopyrrolidin-1-yl 3-(2-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy )ethoxy)ethoxypro panoate), Mal-Tetra-EG-OSu(2,5-dioxopyrrolidin-1-yl 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3- oxo-7,10,13,16-tetraoxa-4-azan onadecan-19-oate), Br-di-EG-OSu(2,5-dioxopyrrolidin-1-yl 3(2-(2-(2-bromoacetamido )ethoxy)ethoxy)propanoate), Py-ds-prp-OSu(2-5-dioxopyrrolidin-1-yl 3-(pyridine-2-yldisulfanyl)propanoate), Py-ds-Prp-OPEP(perfluorophenyl 3-(pyridine -2-yldisulfanyl)propanoate), Py-ds-dmBut-OSu(2,5-dioxopyrrolidin-1-yl 4-methyl-4-(pyridine-2-yldisulfanyl), Py-ds-dmBut-OPF(perfluorophenyl 4- Methyl-4-(pyridine-2-yldisulfanyl)pentanoate), SMCC(N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate), MBS(3-maleimidobenzoic acid N-hydroxysuccinimide ester), SATA(S-(N-succinimidyl)thioacetate) , SPDP((N-succinimidyl 3-(2-pyridyldithio)propionate), SMPT((N-succinimidyloxy carbonyl)-1-methyl-1-(2-pyridyldithio)toluene);
    (3)权利要求1-26中任一项所述的融合蛋白和标记；(3) The fusion protein and marker according to any one of claims 1-26;
    优选地，所述标记选自氧化铁纳米颗粒、紫外-可见标记、近红外标记、发光基团、磷光基团、磁自旋共振标记、光敏剂、光可裂解部分、螯合中心、重原子、放射性同位素、同位素可检测自旋共振标签，顺磁性部分，发色团或其组合；更优选地，所述标记为氧化铁纳米颗粒。Preferably, the label is selected from iron oxide nanoparticles, UV-visible labels, near-infrared labels, luminescent groups, phosphorescent groups, magnetic spin resonance labels, photosensitizers, photo-cleavable moieties, chelating centers, heavy atoms , radioisotopes, isotope detectable spin resonance tags, paramagnetic moieties, chromophores or combinations thereof; more preferably, the tags are iron oxide nanoparticles.
  35. 根据权利要求34所述的前药，所述嵌合抗原受体包含细胞外结构域、跨膜结构域、共刺激信号传导区域和胞内信号传导结构域，所述细胞外结构域结合的抗原选自：间皮素(MSLN)、B7-H3(CD276)、硫酸软骨素蛋白多糖4(CSPG4)、Muc 16、Claudin 18.2、Claudin 8、NY-ESO-1、CD19、CD22、CD23、骨髓增生性白血病蛋白MPL、CD30、CD32、CD20、CD70、CD99、CD123、CD138、CD179b、CD200R、CD324、Fc受体样5FcRH5、CD171、CS-1(信号传导淋巴细胞活化分子家族7 SLAMF7)；所述跨膜结构域为CD8或CD28的跨膜区；所述共刺激分子选自MHC I类分子，TNF受体蛋白，免疫球蛋白样蛋白，细胞因子受体，整联蛋白，信号传导淋巴细胞活化分子(SLAM蛋白)，激活NK细胞受体，BTLA，Toll配体受体，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)；所述胞内信号传导结构域选自CD3ζ，CD3γ，CD3δ，CD3ε，CD5，CD22，CD79a，CD79b，CD278(“ICOS”)，FcεRI，CD66d，DAP10和DAP12。 The prodrug according to claim 34, the chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, a co-stimulatory signaling domain and an intracellular signaling domain, the antigen bound to 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 7 SLAMF7); described The transmembrane domain is the transmembrane region of CD8 or CD28; the costimulatory molecule is selected from MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation Molecule (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 selection From CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI, CD66d, DAP10 and DAP12.
  36. 根据权利要求34所述的前药，所述嵌合自然杀伤细胞受体(CNK)复合体包含以下组件：The prodrug of claim 34, said chimeric natural killer cell receptor (CNK) complex comprising the following components:
    (1)NK激活受体组件，其至少包含NK细胞激活受体或其功能变体，所述NK细胞激活受体包含：(a)NK细胞激活受体胞外结构域(ED)或其功能变体，(b)NK细胞激活受体跨膜结构域(TMD)或其功能变体，以及(c)NK细胞激活受体胞内结构域(ICD)或其功能变体；任选地，所述NK细胞激活受体胞外结构域或其功能变体、所述NK细胞激活受体跨膜结构域或其功能变体和/或所述NK细胞激活受体胞内结构域或其功能变体之间包含铰链或接头；(1) 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 Include hinges or joints between variants;
    (2)CNK信号转接组件，其至少包含(i)NK细胞信号转换器(adaptor)或其功能变体，所述NK细胞信号转换器包含：(a)NK细胞信号转换器胞外结构域(ED)或其功能变体，(b)NK细胞信号转换器跨膜结构域(TMD)或其功能变体，以及(c)NK细胞信号转换器胞内结构域(ICD)或其功能变体；任选地，所述NK细胞信号转换器胞外结构域或其功能变体、所述NK细胞信号转换器跨膜结构域或其功能变体和/或所述NK细胞信号转换器胞内结构域或其功能变体之间包含铰链或接头；和(2) 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 Optionally, the NK cell signal transducer extracellular domain or its functional variant, the NK cell signal transducer transmembrane domain or its functional variant and/or the NK cell signal transducer cell Contain hinges or linkers between intradomains or functional variants thereof; and
    任选地，所述NK激活受体组件、所述CNK信号转接组件之间包含铰链或接头。Optionally, a hinge or a linker is included between the NK activating receptor component and the CNK signal transfer component.
  37. 根据权利要求34所述的前药，所述嵌合蛋白构建体包含内质网相关降解(ER-associated degradation，ERAD)机制蛋白结合结构域和靶向结构域(Targeting domain)；According to the prodrug of claim 34, the chimeric protein construct includes an endoplasmic reticulum-associated degradation (ER-associated degradation, ERAD) mechanism protein binding domain and a targeting domain (Targeting domain);
    更优选地，所述ERAD机制蛋白结合结构域包含病毒内质网驻留蛋白的跨膜结构域或其功能变体和内质网驻留结构域或其功能变体；More preferably, the ERAD mechanism protein binding domain includes 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;
    更优选地，所述病毒内质网驻留蛋白为腺病毒E3-19K或选自以下的至少一种：HCMV糖蛋白US2、US11、US3、US10、US6、HSV ICP47、CPXV12、BHV UL49.5、EBV BNFL2a、HCMV UL16、UL141、UL142、HIV Nef、HIV Vpu、HHV-7 U21、HHV-8 KK3、HHV-8 KK5、MHV-68 MK3、HTLV-1 p12、Cowpox Virus蛋白CPXV203的其他病毒内质网驻留蛋白；More preferably, the viral endoplasmic reticulum resident protein is adenovirus E3-19K or at least one selected from the following: HCMV glycoprotein US2, US11, US3, US10, US6, HSV ICP47, CPXV12, BHV UL49.5 , EBV BNFL2a, HCMV UL16, UL141, UL142, HIV Nef, HIV Vpu, HHV-7 U21, HHV-8 KK3, HHV-8 KK5, MHV-68 MK3, HTLV-1 p12, Cowpox Virus protein CPXV203 in other viruses Plasma reticulum resident proteins;
    更优选地，前述嵌合蛋白构建体，其进一步包含蛋白降解通路成员(例如E3泛素连接酶、蛋白酶体、溶酶体)结合结构域，可选地，所述蛋白降解通路成员结合结构域与所述ERAD机制蛋白结合结构域连接；More preferably, the aforementioned chimeric protein construct further comprises a protein degradation pathway member (such as E3 ubiquitin ligase, proteasome, lysosome) binding domain, optionally, the protein degradation pathway member binding domain Connected to the ERAD mechanism protein binding domain;
    更优选地，其中所述靶向结构域包含特异性靶向靶蛋白的抗体或其功能片段(例如，Fd、Fv、Fab、Fab’、F(ab’)2、Fv(scFv)、单链抗体(scFv)、纳米抗体(nanobody)、双链抗体、三链抗体和四链抗体)；More preferably, wherein the targeting domain comprises an antibody or functional fragment thereof that specifically targets the target protein (e.g., Fd, Fv, Fab, Fab', F(ab')2, Fv(scFv), single chain Antibodies (scFv), nanobodies (nanobodies), diabodies, tri-chain antibodies and tetra-chain antibodies);
    所述靶蛋白为致病蛋白，可选地，所述致病蛋白是肿瘤相关的蛋白、病毒相关的蛋白、免疫功能相关蛋白(包括免疫抑制性蛋白和免疫激活性蛋白)、自身抗原蛋白、或神经退行性疾病相关的蛋白；The target protein is a pathogenic protein. Alternatively, the pathogenic protein is a tumor-related protein, a virus-related protein, an immune function-related protein (including an immunosuppressive protein and an immune-activating protein), an autoantigen protein, or proteins associated with neurodegenerative diseases;
    所述自身抗原蛋白选自下组：与I型糖尿病相关的自身抗原例如，胰岛细胞抗原(ICA)、胰岛素(IAA)、谷氨酸脱羧酶65(GAD65)，胰岛瘤抗原-2(IA-2)；与类风湿性关节炎(RA)相关的自身抗原(autoantigen)例如，瓜氨酸化蛋白/肽抗体，异核糖核蛋白(heterogeneous nuclear ribonucleoprotein A2/B1),aldolase,alpha-enolase,calreticulin,热激活蛋白(HSP60)、BiP、PGK1、压力诱导磷酸蛋白(stress-induced phosphoprotein 1)、FUSE-BP1/2；与系统性红斑狼疮(SLE)相关的自身抗原例如，脱氧核糖核蛋白，SmD1和SmD3，Clq，疮抗凝物(LA)、心磷脂(CL)、β2糖蛋白I(β2 GP I)、凝血酶原(PT)和磷脂酰丝氨酸(PS)；与系统性硬化症(Systemic Sclerosis，SSc)/硬皮病(scleroderma SD)相关自身抗原例如，Scl-70、SSA、Ro52；与自身免疫性肝病相关的抗原例如，线粒体抗原、Spl00、PML、gp210、p62；与重症肌无力相关的自身抗原例如，乙酰胆碱受体；与中枢神经系统自身免疫疾病(边缘性脑炎、脑脊髓炎、小脑性共济失调)相关的自身抗原例如，电压门控性钾离子通道(VGKC)复合物、电压门控性钙离子通道受体、a-氨基-3-羟基-5-甲基异噁唑-4-丙酸(AMPA)受体、γ氨基丁酸-B(GABAB)受体、甘氨酸受体；与多发性硬化相关的自身抗原例如，髓磷脂碱性蛋白(MBP)、髓磷脂少突胶质细胞糖蛋白(MOG)；与多发性肌炎(PM)和皮肌炎(DM)相关的自身抗原例如，Jo-1、Mi-2、PM-Scl、Ro-52；与麸质敏感性肠病相关的自身抗原例如，肌内膜抗体(EMA)、组织谷氨酰胺转移酶(tTG)；与抗NMDAR抗体脑炎相关的自身抗原例如，N-甲基-D-天冬氨酸受体；与视神经脊髓炎(NMO)相关的自身抗原例如，水通道蛋白4(AQP4)；与生殖相关相关的自身抗原例如，卵巢抗原，精子抗原；The autoantigen protein is selected from the following group: autoantigens related to type I diabetes, for example, islet cell antigen (ICA), insulin (IAA), glutamic acid decarboxylase 65 (GAD65), isletoma antigen-2 (IA- 2); Autoantigens related to rheumatoid arthritis (RA), such as citrullinated protein/peptide antibodies, heterogeneous nuclear ribonucleoprotein A2/B1, aldolase, alpha-enolase, calreticulin, Heat-activated protein (HSP60), BiP, PGK1, stress-induced phosphoprotein 1, FUSE-BP1/2; autoantigens related to systemic lupus erythematosus (SLE) such as deoxyribonucleoprotein, SmD1 and SmD3, Clq, ulcer anticoagulant (LA), cardiolipin (CL), β2 glycoprotein I (β2 GP I), prothrombin (PT), and phosphatidylserine (PS); associated with systemic sclerosis , SSc)/scleroderma (scleroderma SD) related autoantigens such as Scl-70, SSA, Ro52; antigens related to autoimmune liver diseases such as mitochondrial antigen, Spl00, PML, gp210, p62; related to myasthenia gravis Autoantigens such as acetylcholine receptors; autoantigens associated with central nervous system autoimmune diseases (limbic encephalitis, encephalomyelitis, cerebellar ataxia) such as voltage-gated potassium channel (VGKC) complex , voltage-gated calcium channel receptor, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor, gamma-aminobutyric acid-B (GABAB) receptor, glycine Receptors; autoantigens associated with multiple sclerosis such as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG); associated with polymyositis (PM) and dermatomyositis (DM) Relevant autoantigens such as Jo-1, Mi-2, PM-Scl, Ro-52; autoantigens associated with gluten-sensitive enteropathy such as endomysial antibodies (EMA), tissue glutamine transferase ( tTG); autoantigens associated with anti-NMDAR antibody encephalitis, such as N-methyl-D-aspartate receptor; autoantigens associated with neuromyelitis optica (NMO), such as aquaporin 4 (AQP4); Autoantigens related to reproduction, such as ovarian antigens and sperm antigens;
    所述肿瘤相关的蛋白由选自由以下组成的组的致癌基因编码：BCL-2、c-MYC、Ras、HER2、BCR/ABL、ABL1/BCR、TGFB1、TLX1、P53、WNT1、WNT2、WT1、αv-β3、PKCa、ABL、BCL1、CD24、CDK4、EGFR/ERBB-1、HSTF1、INT1/WNT1、INT2、MDM2、MET、MYB、MYC、MYCN、MYCL1、RAFI、NRAS、REL、AKT2、APC、BCL2-ALPHA、BCL2-BETA、BCL3、BCR、BRCA1、BRCA2、CBL、CCND1、CDKN1A、CDKN1C、CDKN2A、CDKN2B、CRK、CRK-II、CSF1R/FMS、DBL、DDOST、PMS-2、PRAD-1、RAF、RHOM-1、RHOM-2、SIS、TAL2、TANI、TIAM1、TSC2、TRK、TSC1、STK11、PTCH、MEN1、MEN2、P57/KIP2、PTEN、HPC1、ATM、XPA/XPG、BCL6、DEK、AKAP13、CDH1、BLM、EWSR1/FLI1、FES、FGF3、FER、FGR、FLI1/ERGB2、FOS、FPS/FES、FRA1、FRA2、FYN、HCK、HEK、HER3/ERBB-2、ERBB-3、HER4/ERBB-4、HST2、INK4A、INK4B、JUN、JUNB、JUND、KIP2、KIT、KRAS2A、KRAS2B、LCK、LYN、MAS、MAX、MCC、MLH1、MOS、MSH2、MYBA、MYBB、NF1、NF2、P53、PDGFB、PIM1、PTC、RBI、RET、ROS1、SKI、SRC1、TALI、TGFBR2、THRA1、THRB、TIAM1、TRK、VAV、VHL、WAF1、WNT2、WT1、YES1、ALK/NPM1、AMI1、AXL、FMS、GIP、GLI、GSP、HOX11、HST、IL3、INT2、KS3、K-SAM、LBC、DCC、DPC4/SMAD4、E-CAD、E2F1/RBAP、ELK1、ELK3、EPH、EPHA1、E2F1、EPHA3、ERG、ETS1、ETS2、LMO-1、LMO-2、L-MYC、LYL1、LYT-10、MDM-2、MLH1、MLL、 MLM、N-MYC、OST、PAX-5、PMS-1、FGF4、FGF6、FANCA、FLI1/ERGB2、FOSL1、FOSL2、GLI、HRAS1、HRX/MLLT1、HRX/MLLT2、KRAS2、MADH4、MASI、MCF2、MLLT1/MLL、MLLT2/HRX、MTG8/RUNX1、MYCLK1、MYH11/CBFB、NFKB2、NOTCH1、NPM1/ALK、NRG/REL、NTRK1、PBX1/TCF3、PML/RARA、PRCA1、RUNX1、RUNX1/CBFA2T1、SET、SHP2、TCF3/PBX1、TNFa、Clusterin、Survivin、ΤΟΕβ、c-fos、c-SRC、雌激素受体基因(estrogen receptor ER-α)、雄激素受体基因(Androgen receptor,AR)、和INT-1；可选地，所述肿瘤相关的蛋白选自下组：Bcl-2家族成员(如：Bcl-2,Bcl-xL and Bcl-w)、VEGF/VEGFR、PDGFRβ、EGFR、EGFR突变体、IGF-1R、HDACs、HER2、MYC、KRAS、AFP,CEA,CA199,雌激素受体(estrogen receptor ER-α)、雄激素受体(Androgen receptor,AR)、酪氨酸激酶(c-ABL、BCR-ABL、BTK、FAK、PTK6、Wee1、TRK跨膜受体)、丝/苏氨酸激酶受体(IRAK4、LRRK2、B-Raf、RIPK2、CDK4/6、CDK7、CDK8、CDK8/19、CDK9、TBK1)、蛋白激酶Ⅱ(CK2)、表观遗传相关蛋白(BRD2、BRD3、BRD4、BRDT、TRIM24、BRD9、PBRM1、SMARCA2、SMARCA4、EP300、EZH2、WDR5)、肾上腺髓质素(ADM)、DPP3；The tumor-associated protein is encoded by an oncogene selected from the group consisting of: BCL-2, c-MYC, Ras, HER2, BCR/ABL, ABL1/BCR, TGFB1, TLX1, P53, WNT1, WNT2, WT1, αv-β3, PKCa, ABL, BCL1, CD24, CDK4, EGFR/ERBB-1, HSTF1, INT1/WNT1, INT2, MDM2, MET, MYB, MYC, MYCN, MYCL1, RAFI, NRAS, REL, AKT2, APC, BCL2-ALPHA, BCL2-BETA, BCL3, BCR, BRCA1, BRCA2, CBL, CCND1, CDKN1A, CDKN1C, CDKN2A, CDKN2B, CRK, CRK-II, CSF1R/FMS, DBL, DDOST, PMS-2, PRAD-1, RAF, RHOM-1, RHOM-2, SIS, TAL2, TANI, TIAM1, TSC2, TRK, TSC1, STK11, PTCH, MEN1, MEN2, P57/KIP2, PTEN, HPC1, ATM, XPA/XPG, BCL6, DEK, AKAP13, CDH1, BLM, EWSR1/FLI1, FES, FGF3, FER, FGR, FLI1/ERGB2, FOS, FPS/FES, FRA1, FRA2, FYN, HCK, HEK, HER3/ERBB-2, ERBB-3, HER4/ ERBB-4, HST2, INK4A, INK4B, JUN, JUNB, JUND, KIP2, KIT, KRAS2A, KRAS2B, LCK, LYN, MAS, MAX, MCC, MLH1, MOS, MSH2, MYBA, MYBB, NF1, NF2, P53, PDGFB, PIM1, PTC, RBI, RET, ROS1, SKI, SRC1, TALI, TGFBR2, THRA1, THRB, TIAM1, TRK, VAV, VHL, WAF1, WNT2, WT1, YES1, ALK/NPM1, AMI1, AXL, FMS, GIP, GLI, GSP, HOX11, HST, IL3, INT2, KS3, K-SAM, LBC, DCC, DPC4/SMAD4, E-CAD, E2F1/RBAP, ELK1, ELK3, EPH, EPHA1, E2F1, EPHA3, ERG, ETS1, ETS2, LMO-1, LMO-2, L-MYC, LYL1, LYT-10, MDM-2, MLH1, MLL, MLM, N-MYC, OST, PAX-5, PMS-1, FGF4, FGF6, FANCA, FLI1/ERGB2, FOSL1, FOSL2, GLI, HRAS1, HRX/MLLT1, HRX/MLLT2, KRAS2, MADH4, MASI, MCF2, MLLT1/MLL, MLLT2/HRX, MTG8/RUNX1, MYCLK1, MYH11/CBFB, NFKB2, NOTCH1, NPM1/ALK, NRG/REL, NTRK1, PBX1/TCF3, PML/RARA, PRCA1, RUNX1, RUNX1/CBFA2T1, SET, SHP2, TCF3/PBX1, TNFa, Clusterin, Survivin, ΤΟΕβ, c-fos, c-SRC, estrogen receptor ER-α, androgen receptor (AR), and INT- 1; Optionally, the tumor-related protein is selected from the following group: Bcl-2 family members (such as: Bcl-2, Bcl-xL and Bcl-w), VEGF/VEGFR, PDGFRβ, EGFR, EGFR mutants, IGF-1R, HDACs, HER2, MYC, KRAS, AFP, CEA, CA199, estrogen receptor (estrogen receptor ER-α), 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, PBRM1, SMARCA2, SMARCA4, EP300, EZH2, WDR5), adrenomedullin (ADM) ,DPP3;
    所述病毒相关蛋白，其选自：HBV表面抗原、HBV衣壳糖蛋白、HBeAg、HBV DNA聚合酶、HBV编码X蛋白(HBx)、HIV Gag protein、HIV Env protein、HIV gp120、HIV-1逆转录酶、HIV gp120、HCV NS3-4A蛋白酶、HCV RNA聚合酶、HCV包膜(envelope)蛋白、EBV DNA聚合酶、EBV EBNA1、冠状病毒RNA合成酶、冠状病毒刺突蛋白、冠状病毒包膜蛋白、冠状病毒膜蛋白、冠状病毒核壳蛋白、RNA依赖性RNA聚合酶(RNA-dependent RNA polymerase,RdRp)，如新冠病毒RNA依赖性RNA聚合酶、疱疹病毒(Herpesviruses)DNA和RNA聚合酶、疱疹病毒衣壳糖蛋白、CMV DNA聚合酶、CMV衣壳糖蛋白、RSV表膜蛋白、RSV衣壳蛋白、RSV RNA聚合酶、流感病毒RNA聚合酶、流感病毒包膜(envelope)蛋白、HPV DNA聚合酶、和HPV衣壳蛋白；The virus-related protein is selected from: HBV surface antigen, HBV capsid glycoprotein, HBeAg, HBV DNA polymerase, HBV encoded X protein (HBx), HIV Gag protein, HIV Env protein, HIV gp120, HIV-1 reverse Recordase, HIV gp120, HCV NS3-4A protease, HCV RNA polymerase, HCV envelope protein, EBV DNA polymerase, EBV EBNA1, coronavirus RNA synthase, coronavirus spike protein, coronavirus envelope protein , coronavirus membrane protein, coronavirus nucleocapsid protein, RNA-dependent RNA polymerase (RdRp), such as new coronavirus RNA-dependent RNA polymerase, herpesviruses DNA and RNA polymerase, herpes Viral capsid glycoprotein, CMV DNA polymerase, CMV capsid glycoprotein, RSV membrane protein, RSV capsid protein, RSV RNA polymerase, influenza virus RNA polymerase, influenza virus envelope protein, HPV DNA polymerization enzymes, and HPV capsid proteins;
    所述免疫功能相关蛋白，其选自：抗原呈递分子(例如MHC I类分子、MHC II类分子、MICA/B分子等)、抗原识别分子(例如TCR、CD123、NKG2D等)、免疫检查点分子(例如PD-1、PD-L1、CTLA4、TIM3,TIGIT,LAG3,A2AR,BTLA,IDO1,IDO2,TDO,KIR,NOX2,VISTA,SIGLEC7,PVR等)、免疫刺激/共刺激分子(例如CD3、CD80/86、CD28等)；The immune function-related protein is selected from: antigen presentation 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.), immunostimulatory/costimulatory molecules (such as CD3, CD80/86, CD28, etc.);
    所述神经系统疾病相关的靶蛋白，其选自：Tau、amyloid-β(Aβ)、α突触核蛋白、亨廷顿蛋白突变体(mutant huntingtin,mHTT)、α-synuclein、TAR RNA结合蛋白(TARDBP)和FUS RNA结合蛋白(FUS)；The target protein related to neurological diseases is selected from: Tau, amyloid-β (Aβ), α-synuclein, mutant huntingtin (mHTT), α-synuclein, TAR RNA binding protein (TARDBP) ) and FUS RNA-binding protein (FUS);
    前述嵌合蛋白构建体，其和至少一个共表达部分连接，所述共表达部分独立地选自：完整的病毒ER驻留糖蛋白(例如，HCMV US2、US3、US11、US10、腺病毒E3-Kl 9、HCMV US6、HSV ICP47)、嵌合抗原受体(CAR)、功能性T细胞受体(TCR)、趋化因子受体和NK细胞激活受体；The aforementioned chimeric protein construct is connected to at least one co-expression part, and the co-expression part is independently selected from: complete viral ER resident glycoprotein (for example, HCMV US2, US3, US11, US10, adenovirus E3- Kl 9, HCMV US6, HSV ICP47), chimeric antigen receptor (CAR), functional T cell receptor (TCR), chemokine receptor and NK cell activating receptor;
    所述趋化因子受体选自：CCR4、CCR5、CCR6、CCR7、CCR9、CCR2b、CXCR1、CXCR2和CXCR4；The chemokine receptor is selected from: CCR4, CCR5, CCR6, CCR7, CCR9, CCR2b, CXCR1, CXCR2 and CXCR4;
    所述NK细胞激活受体包含：(a)NK细胞激活受体胞外结构域(ED)或其功能变体，(b)NK细胞激活受体跨膜结构域(TMD)或其功能变体，以及(c)NK细胞激活受体胞内结构域(ICD)或其功能变体；任选地，所述NK细胞激活受体胞外结构域或其功能变体、所述NK细胞激活受体跨膜结构域或其功能变体和/或所述NK细胞激活受体胞内结构域或其功能变体之间包含铰链或接头；The NK cell activating receptor includes: (a) NK cell activating receptor extracellular domain (ED) or a functional variant thereof, (b) NK cell activating receptor transmembrane domain (TMD) or a functional variant thereof , and (c) 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, the NK cell activating receptor A hinge or joint is included between the body transmembrane domain or a functional variant thereof and/or the intracellular domain of the NK cell activating receptor or a functional variant thereof;
    所述NK细胞激活受体选自NKG2D、NKG2C、NKG2E、NKG2F、NKG2H、CD94、KIR2DL4、KIR2DS1、KIR2DS2、KIR2DS4、KIR3DS1，天然细胞毒性受体、TRAIL、DNAM-1、CD16a、2B4、NTB-A、CRACC以及NKp80。The NK cell activating receptor is selected from NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, natural cytotoxicity receptor, TRAIL, DNAM-1, CD16a, 2B4, NTB-A , CRACC and NKp80.
  38. 根据权利要求1-29中任一项所述的融合蛋白、权利要求30所述的核酸、权利要求31所述的载体、权利要求32所述的细胞和/或权利要求34-37中任一项所述的前药在制备用于诊断、治疗或预防癌症或病毒感染类疾病的药物或试剂盒中的用途；The fusion protein according to any one of claims 1 to 29, the nucleic acid according to claim 30, the vector according to claim 31, the cell according to claim 32 and/or any one of claims 34 to 37 The use of the prodrugs described in the item in the preparation of drugs or kits for diagnosing, treating or preventing cancer or viral infection diseases;
    优选地，所述癌症选自白血病、成人晚期癌症、胰腺癌、不可切除性胰腺癌、结肠直肠癌、转移性结肠直肠癌、卵巢癌、三阴性乳腺癌、造血/淋巴癌、结肠癌肝转移、小细胞肺癌、非小细胞肺癌、B细胞淋巴瘤、复发性或难治性B细胞淋巴瘤、滤泡性淋巴瘤、套细胞淋巴瘤、弥漫性大细胞淋巴瘤、复发性或难治性弥漫性大细胞淋巴瘤、间变性大细胞淋巴瘤、原发性纵隔B细胞淋巴瘤、复发性纵隔大B细胞淋巴瘤、难治性纵隔大B细胞淋巴瘤、大B细胞淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、复发性或难治性非霍奇金淋巴瘤、难治性侵袭性非霍奇金淋巴瘤、B细胞非霍奇金淋巴瘤、难治性非霍奇金淋巴瘤、结肠直肠上皮癌、胃癌、胰腺癌、三阴性浸润性乳腺癌、肾细胞癌、肺鳞状细胞癌、肝细胞癌、尿路上皮癌、白血病、B细胞白血病、B细胞急性淋巴细胞白血病、B细胞急性成淋巴细胞白血病、成人急性成淋巴细胞白血病、B细胞幼淋巴细胞白血病、儿童急性成淋巴细胞白血病、难治性儿童急性成淋巴细胞白血病、急性白血病、急性成淋巴细胞白血病、急性淋巴细胞白血病、幼淋巴细胞白血病、慢性淋巴细胞白血病、急性髓性白血病、复发性浆细胞骨髓瘤、难治性浆细胞骨髓瘤、多发性骨髓瘤、复发性或难治性多发性骨髓瘤、骨多发性骨髓瘤、脑恶性神经胶质瘤、骨髓增生异常综合征、EGFR阳性结肠直肠癌、多形性胶质母细胞瘤、赘生物、母细胞性浆细胞样树突状细胞肿瘤、肝转移、实体瘤、晚期实体瘤、间皮素阳性肿瘤、血液恶性肿瘤及其他晚期恶性肿瘤；Preferably, the cancer is selected from the group consisting of leukemia, adult advanced cancer, pancreatic cancer, unresectable pancreatic cancer, colorectal cancer, metastatic colorectal cancer, ovarian cancer, triple negative breast cancer, hematopoietic/lymphoid 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, relapsed mediastinal large B-cell lymphoma, refractory mediastinal large B-cell lymphoma, large B-cell lymphoma, Hodge Golden lymphoma, non-Hodgkin lymphoma, relapsed or refractory non-Hodgkin lymphoma, refractory aggressive non-Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, refractory non-Hodgkin lymphoma Chikin lymphoma, colorectal cancer, gastric cancer, pancreatic cancer, triple-negative invasive breast cancer, renal cell carcinoma, lung squamous cell carcinoma, hepatocellular carcinoma, urothelial carcinoma, leukemia, B-cell leukemia, B-cell acute Lymphocytic leukemia, B-cell acute lymphoblastic leukemia, adult acute lymphoblastic leukemia, B-cell prolymphoblastic leukemia, childhood acute lymphoblastic leukemia, refractory childhood acute lymphoblastic leukemia, acute leukemia, acute lymphoblastic leukemia Leukemia, acute lymphoblastic leukemia, prolymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, relapsed plasma cell myeloma, refractory plasma cell myeloma, multiple myeloma, relapsed or refractory multiple myeloma Myeloma, bone multiple myeloma, brain malignant glioma, myelodysplastic syndrome, EGFR-positive colorectal cancer, glioblastoma multiforme, neoplasia, blastic plasmacytoid dendritic cells Tumors, liver metastases, solid tumors, advanced solid tumors, mesothelin-positive tumors, hematological malignancies and other advanced malignancies;
    优选地，所述病毒感染类疾病包括呼吸道病毒性疾病、胃肠道病毒性疾病、肝脏病毒性疾病、皮肤和黏膜病毒性疾病、眼病毒性疾病、中枢神经系统病毒性疾病、淋巴细胞性病毒性疾病、虫传病毒性疾病、慢病毒感染疾 病等；Preferably, the 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, and lymphocytic viral diseases. Diseases, insect-borne viral diseases, lentiviral infectious diseases Disease, etc.;
    更优选地，呼吸道病毒性疾病包括鼻病毒、腺病毒、呼吸道合胞病毒、副流感病毒和冠状病毒等的感染；流行性感冒；流行性腮腺炎等；More preferably, respiratory viral diseases include infections with rhinovirus, adenovirus, respiratory syncytial virus, parainfluenza virus, coronavirus, etc.; influenza; mumps, etc.;
    更优选地，胃肠道病毒性疾病包括脊髓灰质炎；库克萨基病毒感染；ECHO病毒感染；病毒性胃肠炎：包括轮状病毒性胃肠炎、诺瓦克病毒性胃肠炎、腺病毒性胃肠炎、星状病毒性胃肠炎、冠状病毒性胃肠炎和杯状病毒性胃肠炎等；More preferably, 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.;
    更优选地，肝脏病毒性疾病包括甲型病毒性肝炎、乙型病毒性肝炎、丙型病毒性肝炎、丁型病毒性肝炎、戊型病毒性肝炎、EB病毒性肝炎和巨细胞病毒性肝炎等；More preferably, liver viral diseases include hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, Epstein-Barr virus hepatitis, cytomegalovirus hepatitis, etc. ;
    更优选地，皮肤和黏膜病毒性疾病包括麻疹、风疹、幼儿急疹、水痘及带状疱疹、天花、单纯疱疹病毒感染、狂犬病和口蹄疫等；More preferably, skin and mucosal viral diseases include measles, rubella, exanthema, chickenpox and herpes zoster, smallpox, herpes simplex virus infection, rabies, foot and mouth disease, etc.;
    更优选地，眼病毒性疾病包括流行性角膜结膜炎、滤泡性结膜炎和疱疹性角膜结膜炎等；More preferably, the ocular viral diseases include epidemic keratoconjunctivitis, follicular conjunctivitis, herpetic keratoconjunctivitis, etc.;
    更优选地，中枢神经系统病毒性疾病包括流行性乙型脑炎、西方马脑炎、东方马脑炎、圣路易脑炎、委内瑞拉马脑炎、墨累山谷脑炎、加利福尼亚脑炎、森林脑炎和淋巴细胞脉络丛脑膜炎等；More preferably, the viral diseases of the central nervous system include Japanese encephalitis, Western equine encephalitis, Eastern equine encephalitis, St. Louis encephalitis, Venezuelan equine encephalitis, Murray Valley encephalitis, California encephalitis, forest brain inflammation and lymphocytic choriomeningitis, etc.;
    更优选地，淋巴细胞性病毒性疾病包括传染性单核细胞增多症、巨细胞病毒感染和获得性免疫缺陷综合征等；More preferably, lymphocytic viral diseases include infectious mononucleosis, cytomegalovirus infection, acquired immunodeficiency syndrome, etc.;
    更优选地，虫传病毒性疾病包括病毒性出血热：包括流行性出血热、黄热病、克里米亚－刚果出血热、裂谷热、阿根廷出血热、玻利维亚出血热、拉萨热、鄂木斯克出血热、马尔堡病和埃波拉出血热等；登革热和登革出血热；西尼罗热；科罗拉多蜱传热；白蛉热等；More preferably, the insect-borne viral diseases include viral hemorrhagic fevers: including epidemic hemorrhagic fever, yellow fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever, Hubei fever Muscovite hemorrhagic fever, Marburg disease, Ebola hemorrhagic fever, etc.; Dengue fever and dengue hemorrhagic fever; West Nile fever; Colorado tick-borne fever; sand fly fever, etc.;
    更优选地，慢病毒感染疾病包括亚急性硬化性全脑炎、库鲁病、进行性多灶性白质脑病和亚急性海绵样脑病(皮质纹状体脊髓变性)。 More preferably, lentiviral infectious diseases include subacute sclerosing panencephalitis, kuru disease, progressive multifocal leukoencephalopathy and subacute spongiform encephalopathy (corticostriatal spinal degeneration).
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