Fusion protein of targeted cell membrane receptor protein and application thereof

Abstract

The invention provides fusion protein of targeted cell membrane receptor protein and application of the fusion protein. The fusion protein of the target cell membrane receptor protein can be used for promoting the endocytosis of the cell membrane receptor protein of a clathrin-dependent endocytosis mechanism (CDE). The fusion protein of the target cell membrane receptor protein can be used for promoting the endocytosis of the cell membrane receptor protein of the clathrin-dependent endocytosis mechanism (CDE).

Description

Fusion protein targeting cell membrane receptor protein and its application Technical Field

[0001] The present disclosure relates to the field of therapy, and more particularly, to a fusion protein targeting a cell membrane receptor protein and applications thereof. Background Art

[0002] Endocytosis is a mechanism by which small molecules or substrates are transferred into cells through engulfment of the cell membrane during cellular processes. Endocytosis is generally divided into phagocytosis and pinocytosis. Phagocytosis refers to the internalization of larger particles (0.5–10 μm in diameter), such as pathogens. Pinocytosis involves the internalization of various lipids via small endocytic vesicles. Internalization of pathogens or ligands occurs by binding to the cell membrane, through receptor-dependent or receptor-independent mechanisms, to form endosomes or endocytic vesicles.

[0003] The binding of membrane receptors to extracellular molecules is responsible for transmitting extracellular signals into the cell. Studies have shown that some membrane receptors and ligands form complexes that are internalized and translocated to the cytoplasm or nucleus for signal transmission or degradation via a clathrin-dependent endocytosis mechanism (Aguilar RC et al., 2005). For example, T cell receptors (TCRs), receptor tyrosine kinases (RTKs), and G-protein coupled receptors (GPCRs) can be internalized by directly binding to the adaptor proteins AP2, epsin, or intersectin via a clathrin-dependent endocytosis mechanism (Kourouniotis G et al., 2016; Poole DP et al., 2016).

[0004] The T cell (antigen) receptor (TCR) is a heterodimeric protein located on the surface of T cells. It is the molecule that specifically recognizes antigens and mediates immune responses. Due to the complex V(D)J rearrangement mechanism during TCR cell development, the TCR gene sequence is extremely diverse. Therefore, the TCR is highly diverse and one of the most polymorphic regions in the human genome. The TCR is composed of two polypeptide chains (α / β or γ / δ), each of which is divided into a constant region and a variable region. Each chain contains three highly variable regions: CDR1 (complementarity-determining regions, CDR2, and CDR3). CDR1 and CDR2 are relatively conserved and are responsible for MHC recognition. CDR3 is the region of the TCR that directly contacts the antigen peptide and plays a decisive role in the interaction between the TCR and the antigen peptide-MHC complex. CDR3 is the most variable region and largely determines the diversity of the TCR.

[0005] In resting T cells, the TCR is continuously internalized and re-expressed on the cell surface. However, when the TCR binds to pMHC, forming the pMHC / TCR / CD3 immune complex synapse, TCR internalization rapidly increases and downregulates cell surface expression, reducing recycling and accelerating TCR degradation. This, in turn, activates signaling pathways within the T cell, stimulating T cell proliferation or cytotoxicity. During TCR activation, both the bound TCR and surrounding unbound TCRs are internalized through clathrin-dependent or -independent endocytosis. The internalized bound TCR / CD3 complex is degraded, but the majority of surrounding TCR / CD3 complexes recirculate and migrate to the immunological synapse on the cell membrane.

[0006] CD3 antibody stimulation can also lead to TCR endocytosis, a process also mediated by Clathrin 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.).

[0007] VXT is a trimerization domain derived from the trimerization domain of human type XV collagen. Collagen molecules are the main components of the extracellular matrix and play an important role in cell adhesion, motility, migration, and other activities. The XVT structure is derived from the trimerization domain of human type XV or XVIII collagen. This domain contains four β-sheets and α-helices, forming a triple helical structure.

[0008] Summary of the Invention

[0009] The present disclosure provides a method for designing and expressing a recombinant protein comprising a TCR-targeting domain and a trimerization-promoting domain to target and bind to the TCR structure on the surface of T cells, thereby achieving TCR structure aggregation and forming an immune synapse, thereby inducing endocytosis of the TCR structure, and achieving the functions of targeted T cell-directed labeling, drug and nucleic acid delivery, or detection or treatment.

[0010] The present disclosure also provides a method for designing and expressing a recombinant protein comprising a CD3-targeting domain and a trimerization-promoting domain to target and bind to CD3 on the surface of T cells, thereby achieving CD3 aggregation, inducing CD3 endocytosis, and achieving the functions of targeted T cell-directed labeling, drug and nucleic acid delivery, or detection or treatment.

[0011] CD5 is a member of the scavenger receptor family that is constantly expressed on the surface of T cells and some B1a class B cells. When T cells and B cells are treated with anti-CD5 mAb, CD5 can be rapidly internalized (Xianghuai Lu et al., 2002). CD5-targeted LNPs 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. 2022 Jan 7; 375(6576): 91-96.).

[0012] The present disclosure also provides a method for designing and expressing a recombinant protein comprising a CD5-targeting domain and a trimerization-promoting domain to target and bind to CD5 on the surface of T cells, thereby achieving CD5 aggregation, inducing CD5 endocytosis, and achieving the functions of targeted T cell-directed labeling, drug and nucleic acid delivery, or detection or treatment.

[0013] CD7 is a 40 kDa single-domain transmembrane glycoprotein from the Ig superfamily, densely expressed on the surfaces of mature human T cells and NK cells, as well as their precursors (BF Haynes et al., 1979, H Rabinowich et al., 1994). As a T cell co-stimulator, CD7 plays a crucial role in T cell activation by binding to its ligand, K12 / SECTM (Lyman SD et al., 2000). CD7 is highly expressed on all T cell acute lymphoblastic leukemia cells. Because CD7 is rapidly internalized after binding to its ligand or antibody, rather than being cleaved and lost from the cell surface, it is an important target for ADCs used to treat T lymphoblastic leukemia.

[0014] The present disclosure also provides a method for designing and expressing a recombinant protein comprising a CD7-targeting domain and a trimerization-promoting domain to target and bind to CD7 on the surface of T cells, thereby achieving CD7 aggregation, inducing CD7 endocytosis, and achieving the functions of targeted T cell-directed labeling, drug and nucleic acid delivery, or detection or treatment.

[0015] The 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) (Blood 63(6):1424-1433(1984)), but is not found on hematopoietic stem cells, pro-B cells, normal plasma cells, or other normal tissues (Tedder et al. J. Immunol. 135(2):973-979(1985)). CD20 is thought to regulate an early step in the activation process 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)).

[0016] The present disclosure also provides a method for designing and expressing a recombinant protein comprising a CD20-targeting domain and a trimerization-promoting domain to target and bind to CD20 on the surface of T cells, thereby achieving CD20 aggregation and inducing CD20 endocytosis, thereby achieving the functions of targeted T cell-directed labeling, drug and nucleic acid delivery, or detection or treatment.

[0017] 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 predominant form of CD22 in humans is CD22β, which contains seven immunoglobulin superfamily domains in the extracellular domain ( FIG. 1 ) (Wilson, GL et al., J. Exp. Med. 173: 137-146 (1991)). A 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)).

[0018] The present disclosure also provides a method for designing and expressing a recombinant protein comprising a CD22-targeting domain and a trimerization-promoting domain to target and bind to CD22 on the surface of T cells, thereby achieving CD22 aggregation, inducing CD22 endocytosis, and achieving the functions of targeted T cell-directed labeling, drug and nucleic acid delivery, or detection or treatment.

[0019] Fc receptors are proteins found on the surface of antigen-presenting cells, including B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, human platelets, and mast cells, that help protect the immune system. The Fc receptor gets its name from its binding specificity for a portion of an antibody known as the Fc (fragment crystallizable) region. Fc receptors bind to antibodies attached to infected cells or invading pathogens. Fc receptors stimulate phagocytes or cytotoxic cells to destroy microorganisms or infected cells through antibody-mediated phagocytosis or antibody-dependent cellular cytotoxicity. Some viruses (such as flaviviruses) can increase viral invasion of cells through the mechanism of antibody-dependent enhancement of infection (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).

[0020] The present disclosure also provides a method for designing and expressing a recombinant protein comprising a domain targeting an Fc receptor and a trimerization-promoting domain, which targets and binds to Fc receptors on the surface of T cells, thereby achieving Fc receptor aggregation, inducing Fc receptor endocytosis, and achieving the functions of targeted T cell-directed labeling, drug and nucleic acid delivery, or detection or treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 shows the basic structure of the fusion protein and its possible protein structure forms, wherein Figure 1A is a basic structural diagram of the fusion protein, including a targeting domain, a linker, and a trimer domain; Figure 1B is a basic structural diagram of a TCR-targeting fusion protein (BMA-STII-VXT), including a TCR-targeting single-chain antibody fragment (ScFv), a Strep tag II tag, a glycine-Serine linker, and a collagen trimer domain (VXT); Figure 1C is a basic structural diagram of a CD3-targeting fusion protein (BMC-STII-VXT), including a CD3-targeting single-chain antibody fragment (ScFv), a Strep tag II tag, a glycine-Serine linker, and a collagen trimer domain (VXT); Figure 1D is a basic structural diagram of a CD5-targeting fusion protein (BMD-STII-VXT), including a CD5-targeting single-chain antibody fragment (ScFv), a Strep tag II tag, a glycine-Serine linker, and a collagen trimer domain (VXT). tag II tag, glycine-Serine linker, and collagen trimer domain (VXT); Figure 1E is a basic structural diagram of a fusion protein targeting CD7 (BME-STII-VXT), including a single-chain antibody fragment (ScFv) targeting CD7, a Strep tag II tag, a glycine-Serine linker, and a collagen trimer domain (VXT); Figure 1F is a basic structural diagram of a fusion protein targeting CD4 (BMF-STII-VXT), including a single-chain antibody fragment (ScFv) targeting CD4, a Strep tag II tag, a glycine-Serine linker, and a collagen trimer domain (VXT); Figure 1G is a basic structural diagram of a fusion protein targeting CD20 (BMG-STII-VXT), including a single-chain antibody fragment (ScFv) targeting CD20, a Strep tag II tag, glycine-Serine linker, and collagen trimer domain (VXT); FIG1H is a basic structural diagram of the CD22-targeting fusion protein (BMH-STII-VXT), which includes a CD22-targeting single-chain antibody fragment (ScFv), a Strep tag II tag, a glycine-Serine linker, and a collagen trimer domain (VXT);Figure 1I shows the basic structure of a CD64-targeting fusion protein (BMI-STII-VXT), which includes a CD64-targeting single-chain antibody fragment (ScFv), a Strep tag II tag, a glycine-serine linker, and a collagen trimer domain (VXT). Figure 1J shows possible protein structures of the fusion protein: monomer, dimer (targeting domain forms a heterodimer), dimer (targeting domain forms a homodimer), and trimer. Figure 1K shows the results of SDS-PAGE separation of the TCR-targeting fusion protein, showing that in a reduced state, the expressed protein is a monomer, while in a non-reduced state, the expressed protein is a dimer.

[0022] Figure 2 shows the flow cytometry results of the specific binding of the 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 compete with TCR-αβ antibodies for binding to the TCR on the surface of T cells.

[0023] Figure 3 shows the flow cytometry results of T cells simultaneously labeled with a TCR-targeting fusion protein (BMA-STII-VXT) and a TCR-ab antibody. Figure 3A shows that 95.5% of T cells showed high expression of TCR-αβ. Figure 3B shows that BMA-STII-VXT can completely block TCR-αβ antibodies and compete for binding to T cells; Figure 3C shows that BMA-STII-VXT can competitively bind to T cells, preventing TCR-αβ antibodies from binding to T cells; Figure 3D shows that BMA-VXT bound to TCR is internalized into T cells, and some TCRs return to the T cell surface after 6 hours.

[0024] FIG4 shows a schematic structural diagram of the connection between the TCR-targeting fusion protein and the LNP containing the nucleic acid.

[0025] FIG5 shows a schematic structural diagram of the connection between the TCR-targeting fusion protein and the LNP carrying the transposon inside.

[0026] FIG6 shows a schematic structural diagram of the connection between the TCR-targeting fusion protein and the LNP encapsulated with chemotherapy drugs. DETAILED DESCRIPTION

[0027] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0028] As used herein, the term "about" refers to a measurable value, such as an amount, duration, etc., and includes variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% from the specified value.

[0029] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be complete immunoglobulins derived from natural or recombinant sources and can be immunoreactive portions of complete immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in the present disclosure can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, nanobodies, Fv, Fab, and F(ab), as well as single-chain antibodies, diabodies, triabodies, tetrabodies, and humanized antibodies.

[0030] 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 of the antibody that binds to an antigen (e.g., one or more CDRs 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 to the same antigen as the antibody from which the antibody fragment was 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, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments may comprise 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 replacing the corresponding region), obtains immunospecific binding (i.e., exhibits at least or at least about 10 7 -10 8 M -1 "Functional fragments" or "analogs of anti-CCR8 antibodies" are fragments or analogs that can prevent or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction. As used herein, functional fragments generally have the same meaning as "antibody fragments" and, with respect to antibodies, can refer to fragments that can prevent or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction, such as Fv, Fab, F(ab')2, etc. "Fv" fragments are dimers (V H -V L In this configuration, the three CDRs of each variable domain interact to determine the V H -V LThe target binding site on the surface of the dimer is the same as that of the intact antibody. The six CDRs together confer the target binding specificity of the intact antibody. However, even a single variable domain (or half of an Fv comprising only three target-specific CDRs) can still have the ability to recognize and bind to the target. With respect to antibody chain polypeptide sequences, the phrase "substantially identical" can be understood as an antibody chain that exhibits at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polypeptide sequence. With respect to nucleic acid sequences, the term can be understood as a nucleotide sequence that exhibits at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference nucleic acid sequence.

[0031] Sequence "identity" or "identity" has an art-recognized meaning, and published techniques can be used to calculate the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions. 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 for measuring the 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)).

[0032] The term "membrane receptor protein" refers to proteins on or within the cell membrane that have specific functions. They 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 monomeric receptors and complex receptors.

[0033] The term "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphoteric compounds, plasmids and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted as including non-plasmids 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 lentiviruses, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc. Examples of non-viral vectors include but are not limited to CRISPR vector systems, Sleeping Beauty transposon systems, etc. "Activation" as used herein refers to the state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector functions. The term "activated T cells" refers in particular to T cells undergoing cell division.

[0034] The present disclosure provides a fusion protein comprising a module that binds to an antigen and a module that promotes trimer formation, wherein the antigen comprises a cell membrane receptor protein capable of achieving clathrin-dependent endocytosis (CDE).

[0035] In some preferred embodiments, the cell membrane receptor protein is selected from one or more of T cell membrane receptor antigens, B cell membrane receptor antigens and monocyte and other antigen presenting cell membrane receptor antigens.

[0036] 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.

[0037] In some preferred embodiments, the B cell membrane receptor antigen is selected from CD20 and CD22.

[0038] In some preferred embodiments, the membrane receptor antigen of the antigen-presenting cell such as monocytes is selected from Fc receptor antigens.

[0039] In some preferred embodiments, the Fc receptor antigen is selected from one or more of Fcα, FcγRI, FcγRII, FcγRIV, FcεRI and Fcα / μR.

[0040] In some preferred embodiments, the Fca is FcaRI, more preferably CD89.

[0041] In some preferred embodiments, FcγRI is CD64; preferably, FcγRII is FcγRIIB2 (CD32).

[0042] In some preferred embodiments, the FcεRI is CD89.

[0043] In some preferred embodiments, the aforementioned components that promote trimer formation are selected from adiponectin or its collagen-like domain, T4 fibrin trimer domain, trimeric structure of human collagen, such as: the amino-terminal NC1 domain of transmembrane collagens XIII, XV, XVII and XVIII, the NC2 domain of fibril-associated collagens IX, XII, XIV, XVI, XIX, XX and XXI, the GCN4 trimerization domain, human lung 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 the complementary heptad repeat region of enveloped virus class I fusion protein.

[0044] In some preferred embodiments, the component that promotes trimer formation is the amino-terminal NC1 domain of XV.

[0045] In some preferred embodiments, the component that promotes trimer formation comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.18, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical.

[0046] In some preferred embodiments, the component promoting trimer formation comprises the amino acid sequence shown in SEQ ID NO. 18 or the amino acid sequence obtained by deleting, adding or substituting 1, 2 or 3 residues in SEQ ID NO: 18.

[0047] In some preferred embodiments, the aforementioned component that binds to the antigen is selected from an antibody or an antigen-binding fragment thereof to the antigen, or a ligand that binds to the membrane receptor protein.

[0048] In some preferred embodiments, the antibody or antigen-binding fragment thereof to the aforementioned TCR antigen comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO.1 or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence SEQ ID NO.2 or any variant thereof, a heavy chain CDR3 selected from the amino acid sequence SEQ ID NO.3 or any variant thereof; and / or a light chain CDR1 selected from the amino acid sequence SEQ ID NO.4 or any variant thereof, a light chain CDR2 selected from the amino acid sequence SEQ ID NO.5 or any variant thereof, and a light chain CDR3 selected from the amino acid sequence SEQ ID NO.6 or any variant thereof.

[0049] In some preferred embodiments, the antibody or antigen-binding fragment thereof for the aforementioned TCR antigen comprises a heavy chain and light chain CDR combination selected from the following:

[0050] The heavy chain CDR1, CDR2 and CDR3 sequences comprise SEQ ID NOs: 1, 2, and 3, respectively, and / or the light chain CDR1, CDR2 and CDR3 sequences comprise SEQ ID NOs: 4, 5, and 6, respectively.

[0051] 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 a light chain variable region selected from the amino acid sequence SEQ ID NO. 8 or any variant thereof.

[0052] In some preferred embodiments, the aforementioned antibody or antigen-binding fragment thereof to the CD3 antigen comprises a heavy chain CDR1 selected from the amino acid sequence of SEQ ID NO.9 or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence of SEQ ID NO.10 or any variant thereof, and a heavy chain CDR3 selected from the amino acid sequence of SEQ ID NO.11 or any variant thereof; and / or a light chain CDR1 selected from the amino acid sequence of SEQ ID NO.12 or any variant thereof, a light chain CDR2 selected from the amino acid sequence of SEQ ID NO.13 or any variant thereof, and a light chain CDR3 selected from the amino acid sequence of SEQ ID NO.14 or any variant thereof.

[0053] In some preferred embodiments, the CD3 antibody or antigen-binding fragment thereof comprises the heavy chain CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NOs: 9, 10, and 11, respectively, and / or comprises the light chain CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NOs: 12, 13, and 14, respectively.

[0054] In some preferred embodiments, the CD3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 15 or any variant thereof, and / or a light chain variable region selected from the amino acid sequence of SEQ ID NO. 16 or any variant thereof.

[0055] In some preferred embodiments, the antibody or antigen-binding fragment thereof to the CD5 antigen comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO.215 or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence SEQ ID NO.216 or any variant thereof, a heavy chain CDR3 selected from the amino acid sequence SEQ ID NO.217 or any variant thereof; and / or a light chain CDR1 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, and a light chain CDR3 selected from the amino acid sequence SEQ ID NO.220 or any variant thereof.

[0056] In some preferred embodiments, the antibody or antigen-binding fragment thereof to the CD5 antigen comprises heavy chain CDR1, CDR2 and CDR3 as shown in amino acid sequences of SEQ ID NOs: 215, 216, and 217, respectively, and / or comprises light chain CDR1, CDR2 and CDR3 as shown in amino acid sequences of SEQ ID NOs: 218, 219, and 220, respectively.

[0057] In some preferred embodiments, the antibody or antigen-binding fragment thereof against 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 a light chain variable region selected from the amino acid sequence SEQ ID NO. 222 or any variant thereof.

[0058] In some preferred embodiments, the antibody or antigen-binding fragment thereof to the CD7 antigen comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO.223 or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence SEQ ID NO.224 or any variant thereof, a heavy chain CDR3 selected from the amino acid sequence SEQ ID NO.225 or any variant thereof; and / or a light chain CDR1 selected from the amino acid sequence SEQ ID NO.226 or any variant thereof, a light chain CDR2 selected from the amino acid sequence SEQ ID NO.227 or any variant thereof, and a light chain CDR3 selected from the amino acid sequence SEQ ID NO.228 or any variant thereof.

[0059] In some preferred embodiments, the antibody or antigen-binding fragment thereof to the CD7 antigen comprises heavy chain CDR1, CDR2 and CDR3 as shown in amino acid sequences of SEQ ID NOs: 223, 224, and 225, respectively, and / or comprises light chain CDR1, CDR2 and CDR3 as shown in amino acid sequences of SEQ ID NOs: 226, 227, and 228, respectively.

[0060] In some preferred embodiments, the antibody or antigen-binding fragment thereof against 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 a light chain variable region selected from the amino acid sequence SEQ ID NO. 230 or any variant thereof.

[0061] In some preferred embodiments, the antibody or antigen-binding fragment thereof to the CD4 antigen comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO.231 or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence SEQ ID NO.232 or any variant thereof, a heavy chain CDR3 selected from the amino acid sequence SEQ ID NO.233 or any variant thereof; and / or a light chain CDR1 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, and a light chain CDR3 selected from the amino acid sequence SEQ ID NO.236 or any variant thereof.

[0062] In some preferred embodiments, the antibody or antigen-binding fragment thereof to the CD4 antigen comprises the heavy chain CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NOs: 231, 232 and 233, respectively, and / or comprises the light chain CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NOs: 234, 235 and 236, respectively.

[0063] In some preferred embodiments, the antibody or antigen-binding fragment thereof against the CD4 antigen comprises a heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 237 or any variant thereof, and / or a light chain variable region selected from the amino acid sequence of SEQ ID NO. 238 or any variant thereof.

[0064] In some preferred embodiments, the antibody or antigen-binding fragment thereof to the CD20 antigen comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO.239 or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence SEQ ID NO.240 or any variant thereof, and a heavy chain CDR3 selected from the amino acid sequence SEQ ID NO.241 or any variant thereof; and / or a 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, and a light chain CDR3 selected from the amino acid sequence SEQ ID NO.244 or any variant thereof.

[0065] In some preferred embodiments, the antibody or antigen-binding fragment thereof to the CD20 antigen comprises the heavy chain CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NOs: 239, 240, and 241, respectively, and / or comprises the light chain CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NOs: 242, 243, and 244, respectively.

[0066] In some preferred embodiments, the antibody or antigen-binding fragment thereof against the CD20 antigen comprises a heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 245 or any variant thereof, and / or a light chain variable region selected from the amino acid sequence of SEQ ID NO. 246 or any variant thereof.

[0067] In some preferred embodiments, the antibody or antigen-binding fragment thereof to the CD22 antigen comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO.247 or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence SEQ ID NO.248 or any variant thereof, and a heavy chain CDR3 selected from the amino acid sequence SEQ ID NO.249 or any variant thereof; and / or a light chain CDR1 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, and a light chain CDR3 selected from the amino acid sequence SEQ ID NO.252 or any variant thereof.

[0068] In some preferred embodiments, the antibody or antigen-binding fragment thereof to the CD22 antigen comprises the heavy chain CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NOs: 247, 248, and 249, respectively, and / or comprises the light chain CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NOs: 250, 251, and 252, respectively.

[0069] In some preferred embodiments, the antibody or antigen-binding fragment thereof against the CD22 antigen comprises a heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 253 or any variant thereof, and / or a light chain variable region selected from the amino acid sequence of SEQ ID NO. 254 or any variant thereof.

[0070] In some preferred embodiments, the antibody or antigen-binding fragment thereof to the CD64 antigen comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO.255 ​​or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence SEQ ID NO.256 or any variant thereof, a heavy chain CDR3 selected from the amino acid sequence SEQ ID NO.257 or any variant thereof; and / or a 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, and a light chain CDR3 selected from the amino acid sequence SEQ ID NO.260 or any variant thereof.

[0071] In some preferred embodiments, the antibody or antigen-binding fragment thereof against the CD64 antigen comprises heavy chain CDR1, CDR2 and CDR3 as shown in amino acid sequences of SEQ ID NOs: 255, 256, and 257, respectively, and / or comprises light chain CDR1, CDR2 and CDR3 as shown in amino acid sequences of SEQ ID NOs: 258, 259, and 260, respectively.

[0072] In some preferred embodiments, the antibody or antigen-binding fragment thereof against 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 a light chain variable region selected from the amino acid sequence SEQ ID NO. 262 or any variant thereof.

[0073] In some preferred embodiments, the antigen-binding component and the trimer-forming promoting component in the aforementioned fusion protein can be directly connected or connected through a linker L1.

[0074] In some preferred embodiments, the linker is a flexible linker.

[0075] In some preferred embodiments, the fusion protein further comprises a purification tag; the purification tag is located at the N-terminus, C-terminus, or between the antigen-binding component and the trimer-forming promoting component of the fusion protein.

[0076] In some preferred embodiments, the purification tag is between the component that binds to the antigen and the component that promotes trimer formation.

[0077] In some preferred embodiments, the purification tag is selected from the group consisting of a GST tag, a His tag, a Myc tag, an E tag, a Strep tag, and a HA tag.

[0078] In some preferred embodiments, the Strep tag is a STII tag, and its amino acid sequence is shown in SEQ ID NO.17.

[0079] 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), GGGGSGGGGSGGGGS (SEQ ID NO.24), GGGGSGGGGSGGGG (SEQ ID NO.25), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO.26), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO.27), GSGSGSGS (SEQ ID NO.28), GGSGSGSG (SEQ ID NO.29), GGSGSG (SEQ ID NO.30), and GGSG (SEQ ID NO.31).

[0080] 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).

[0081] In some preferred embodiments, the fusion protein comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO:36, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; preferably, the amino acid sequence is the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence obtained by deleting, adding or substituting 1, 2 or 3 residues in SEQ ID NO:36.

[0082] In another aspect, the present disclosure provides a nucleic acid encoding the aforementioned fusion protein.

[0083] In another aspect, the present disclosure provides a vector comprising the aforementioned nucleic acid.

[0084] In another aspect, the present disclosure provides a cell comprising the aforementioned nucleic acid or vector.

[0085] 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.

[0086] In another aspect, the present disclosure provides a prodrug comprising any one selected from the following:

[0087] (1) the aforementioned fusion proteins and LNPs;

[0088] (2) the aforementioned fusion protein and cytotoxin, wherein the fusion protein and the cytotoxin are connected via a linker unit; or

[0089] (3) The aforementioned fusion protein and tag.

[0090] In a preferred embodiment, the fusion protein and the LNP are connected via a linker L2; more preferably, the linker L2 is a polypeptide having a cysteine ​​residue added to the C-terminus of a flexible polypeptide; 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), GGGGSGGGGSGGGGS (SEQ ID NO.25), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO.26), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO.27), GSGSGSGS (SEQ ID NO.28), GGSGSGSG (SEQ ID NO.29), GGSGSG (SEQ ID NO.30), GGSGSG (SEQ ID NO.31), GGSGSG (SEQ ID NO.32), GGSGSG (SEQ ID NO.33), GGSGSG (SEQ ID NO.34), GGSGSG (SEQ ID NO.35), GGGGSGGGSGGGGSGGGGS (SEQ ID NO.36), GGGGSGGGGSGGGGS (SEQ ID NO.37), GGSGSGS (SEQ ID NO.38), GGSGSGS (SEQ ID NO.39), GGSGSGS (SEQ ID NO.40), GGSGSGS (SEQ ID NO.41), GGSGSGS (SEQ ID NO.42), GGSGSGS (SEQ ID NO.43), GGSGS NO.30), GGSG (SEQ ID NO.31).

[0091] In a preferred embodiment, the linker L2 is selected from the group consisting of GGGSC (SEQ ID NO. 32), GGGGSGGGGSC (SEQ ID NO. 33), GGGGSGGGGSGGGGSGGGGSC (SEQ ID NO. 34) and GGGGSGGGGSGGGSGGGGSC (SEQ ID NO. 35).

[0092] 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, the small molecule compound is a chemotherapy drug; preferably, the chemotherapy drug is a cytotoxin.

[0093] In a preferred embodiment, the nucleic acid having biological activity is selected from one or more of a nucleic acid encoding a chimeric antigen receptor (CAR), a nucleic acid encoding a CNK complex, a nucleic acid encoding a pro-apoptotic protein, and a nucleic acid encoding a chimeric protein construct.

[0094] In a preferred embodiment, the nucleic acid having biological activity 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.

[0095] In a preferred embodiment, the target gene is selected from one or more of the aforementioned chimeric protein receptor (CAR), chimeric natural killer cell receptor (CNK) complex or chimeric protein construct. 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(1 Suppl): S15-S20. doi: 10.1016 / j.bbmt.2010.09.019).

[0096] In a preferred embodiment, the aforementioned chimeric antigen receptor (CAR) comprises an extracellular domain, a transmembrane domain, a costimulatory signaling region, and an intracellular signaling domain, wherein the antigen bound by the extracellular domain is selected from the group consisting of: mesothelin (MSLN), B7-H3 (CD276), chondroitin sulfate proteoglycan 4 (CSPG4), Muc 16, Claudin 18.2, Claudin 8, NY-ESO-1, CD19, CD22, CD23, myeloproliferative leukemia protein 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); 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 molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, 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.

[0097] In a preferred embodiment, the present disclosure provides a multifunctional complex comprising the following components:

[0098] (1) An NK cell activating receptor component, comprising at least an NK cell activating receptor or a functional variant thereof, wherein the NK cell activating receptor comprises: (a) an NK cell activating receptor extracellular domain (ED) or a functional variant thereof, (b) an NK cell activating receptor transmembrane domain (TMD) or a functional variant thereof, and (c) an NK cell activating receptor intracellular domain (ICD) or a functional variant thereof; optionally, a hinge or linker is contained between the NK cell activating receptor extracellular domain or a functional variant thereof, the NK cell activating receptor transmembrane domain or a functional variant thereof, and / or the NK cell activating receptor intracellular domain or a functional variant thereof;

[0099] (2) A CNK signal transduction component, comprising at least (i) an NK cell signal transducer (adaptor) or a functional variant thereof, wherein 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, a hinge or linker is contained between the NK cell signal transducer extracellular domain or a functional variant thereof, 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; and

[0100] Optionally, a hinge or linker is included between the NK activating receptor component and the CNK signal transduction component.

[0101] In some embodiments, the NK cell activating receptor in the NK activating receptor assembly 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; preferably, the natural cytotoxicity receptor is selected from NKp46, NKp44 and NKp30.

[0102] In some preferred embodiments, the NK cell activating receptor is a NK cell activating receptor of mammalian origin; preferably, the mammal is selected from humans, primates, mice, horses, cows, sheep, goats, cats, pigs, dogs, llamas, alpacas, elephants, squirrels, and guinea pigs.

[0103] 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.

[0104] In some preferred embodiments, the NK cell activating receptor is a human NK cell activating receptor; preferably, the NK cell activating receptor is a recombinant NK cell activating receptor comprising different human NK cell activating receptor domains.

[0105] In some preferred embodiments, the NK cell activating receptor is a murine NK cell activating receptor; preferably, the NK cell activating receptor is a recombinant NK cell activating receptor comprising different murine NK cell activating receptor domains.

[0106] 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.

[0107] In some preferred embodiments, the extracellular domain of the NK cell activating receptor is the extracellular domain of a human or mouse NK cell activating receptor.

[0108] In some preferred embodiments, the transmembrane domain of the NK cell activating receptor is a transmembrane domain of a human or mouse NK cell activating receptor.

[0109] In some preferred embodiments, the intracellular domain of the NK cell activating receptor is the intracellular domain of a human or mouse NK cell activating receptor.

[0110] In some preferred embodiments, the functional variant of the NK cell activating receptor is selected from a mutant of the NK cell activating receptor, a wild-type fusion protein, or a fusion protein of the wild-type and mutant forms. In some preferred embodiments, the extracellular domain of human NKG2D comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 37, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity; the amino acid sequence of the extracellular domain of human NKG2D is as shown in SEQ ID NO. 37.

[0111] In some preferred embodiments, the full-length sequence of human NKG2D comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.38, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKG2D is as shown in SEQ ID NO.38.

[0112] In some preferred embodiments, the extracellular domain of mouse NKG2D comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.39, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the extracellular domain of mouse NKG2D is as shown in SEQ ID NO.39.

[0113] In some preferred embodiments, the full-length sequence of mouse NKG2D comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO:40, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of mouse NKG2D is as shown in SEQ ID NO.40.

[0114] In some preferred embodiments, the full-length sequence of human-mouse recombinant NKG2D comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO:41, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human-mouse recombinant NKG2D is as shown in SEQ ID NO.41.

[0115] In some preferred embodiments, the full-length sequence of human NKG2C comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.42, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKG2C is as shown in SEQ ID NO.42.

[0116] In some preferred embodiments, the full-length sequence of human NKG2E comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 43, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKG2E is as shown in SEQ ID NO.43.

[0117] In some preferred embodiments, the full-length sequence of human NKG2F comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 44, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKG2F is as shown in SEQ ID NO.44.

[0118] In some preferred embodiments, the full-length sequence of human CD94 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 45, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of human CD94 is shown in SEQ ID NO. 45.

[0119] In some preferred embodiments, the full-length sequence of human KIR2DL4 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 46, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human KIR2DL4 is as shown in SEQ ID NO.46.

[0120] In some preferred embodiments, the full-length sequence of human KIR2DS1 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 47, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human KIR2DS1 is as shown in SEQ ID NO.47.

[0121] In some preferred embodiments, the full-length sequence of human KIR2DS2 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 48, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human KIR2DS2 is as shown in SEQ ID NO.48.

[0122] In some preferred embodiments, the full-length sequence of human KIR2DS4 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 49, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human KIR2DS4 is as shown in SEQ ID NO.49.

[0123] In some preferred embodiments, the full-length sequence of human KIR3DS1 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 50, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human KIR3DS1 is as shown in SEQ ID NO.50.

[0124] In some preferred embodiments, the full-length sequence of human NKp46 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.51, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKp46 is as shown in SEQ ID NO.51.

[0125] In some preferred embodiments, the full-length sequence of human NKp44 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.52, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKp44 is as shown in SEQ ID NO.52.

[0126] In some preferred embodiments, the full-length sequence of human NKp30 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.53, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKp30 is as shown in SEQ ID NO.53.

[0127] In some preferred embodiments, the full-length sequence of human DNAM1 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 54, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human DNAM1 is as shown in SEQ ID NO.54.

[0128] In some preferred embodiments, the full-length sequence of human TRAIL comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 55, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human TRAIL is as shown in SEQ ID NO.55.

[0129] In some preferred embodiments, the full-length sequence of human CD16a comprises an amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO: 56, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length human CD16a is as shown in SEQ ID NO. 56.

[0130] In some preferred embodiments, the full-length sequence of human 2B4 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 57, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human 2B4 is shown in SEQ ID NO. 57.

[0131] In some preferred embodiments, the full-length sequence of human NTB-A comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 58, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NTB-A is as shown in SEQ ID NO. 58.

[0132] In some preferred embodiments, the full-length sequence of human CRACC comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 59, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human CRACC is shown in SEQ ID NO. 59.

[0133] In some preferred embodiments, the full-length sequence of human NKp80 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 60, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKp80 is as shown in SEQ ID NO.60.

[0134] In some embodiments, the NK cell signal converter in the CNK signal converter component is DAP10 or DAP12.

[0135] In some preferred embodiments, the NK cell signal transducer is a NK cell signal transducer of mammalian origin; preferably, the mammal is selected from humans, primates, mice, horses, cows, sheep, goats, cats, pigs, dogs, llamas, alpacas, elephants, squirrels, and guinea pigs.

[0136] 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.

[0137] In some preferred embodiments, the NK cell signal transducer is a human NK cell signal transducer; preferably, the NK cell signal transducer is a recombinant NK cell signal transducer comprising different human NK cell signal transducer domains.

[0138] In some preferred embodiments, the NK cell signal transducer is a murine NK cell signal transducer; preferably, the NK cell signal transducer is a recombinant NK cell signal transducer comprising different murine NK cell signal transducer domains.

[0139] 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.

[0140] In some preferred embodiments, the extracellular domain of the NK cell signal transducer is the extracellular domain of a human or mouse NK cell signal transducer.

[0141] In some preferred embodiments, the transmembrane domain of the NK cell signal transducer is a transmembrane domain of a human or mouse NK cell signal transducer.

[0142] In some preferred embodiments, the intracellular domain of the NK cell signal transducer is the intracellular domain of a human or mouse NK cell signal transducer;

[0143] In some preferred embodiments, the CNK cell signal transducer functional variant is selected from a DAP10 mutant or a DAP12 mutant, or a fusion protein of DAP10 and DAP12, or a fusion protein of wild-type DAP10 or DAP12 and a mutant DAP10 or DAP12.

[0144] In some preferred embodiments, the CNK signal transduction component further comprises (ii) an immunoreceptor activation signaling domain (ITAM) and / or (iii) a T cell co-stimulatory signaling domain.

[0145] In some preferred embodiments, a hinge or linker is contained between the NK cell signal transducer or a functional variant thereof, the immunoreceptor activation signaling domain (ITAM) and / or the T cell co-stimulatory signaling domain; preferably, the NK cell signal transducer or a functional variant thereof is fused with the immunoreceptor activation signaling domain (ITAM) domain.

[0146] In some preferred embodiments, the immunoreceptor activation signaling domain (ITAM) is derived from the intracellular activation signaling domain of an immunoreceptor; preferably, the immunoreceptor is selected from TCRζ, CD2, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, FcRγ, CD66d, FcαRI, FcγRI, FcγRII, FcγRIII, Dectin-1, CLEC-1, CD72, CD79A, CD79B; preferably, the immunoreceptor activation signaling domain (ITAM) is fused with an NK cell signal transducer or a functional variant thereof; preferably, the immunoreceptor is CD3ζ.

[0147] In some preferred embodiments, the T cell co-stimulatory signaling domain is derived from the intracellular signaling domain of a co-stimulatory molecule; preferably, the co-stimulatory molecule is selected from MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD16, CD27, CD28, CD30, CD40, CD38, CD35, CD79A, CD79B, CDS, ICAM-1, LFA-1, (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFF R, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c , ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, NCR, DAP10, DAP12, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2 B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM( SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligand that specifically binds to CD83, CARD11, FcRa, FcRp, FcRy, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, Wnt, OX40, ROR2, Ryk, SLAMF1, Slp76, pTa, TCRa, TCRp, TRIM, ZAP70, PTCH2.

[0148] In some preferred embodiments, the full-length sequence of human DAP10 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.61, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human DAP10 is as shown in SEQ ID NO.61.

[0149] In some preferred embodiments, the full-length sequence of human DAP10 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.62, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human DAP10 is as shown in SEQ ID NO.62.

[0150] In some preferred embodiments, the transmembrane domain of human DAP10 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.63, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the transmembrane domain of human DAP10 is as shown in SEQ ID NO.63.

[0151] In some preferred embodiments, the full-length sequence of human DAP12 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.64, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human DAP12 is as shown in SEQ ID NO.64.

[0152] In some preferred embodiments, the transmembrane domain of human DAP12 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.65, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the transmembrane domain of human DAP12 is as shown in SEQ ID NO.65.

[0153] In some preferred embodiments, the transmembrane domain fusion protein of human DAP10 and human DAP12 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.66, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequences of the transmembrane domains of human DAP10 and human DAP12 are as shown in SEQ ID NO.66.

[0154] In some preferred embodiments, the human DAP10-DAP12 fusion protein sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.67, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP10-DAP12 fusion protein sequence is as shown in SEQ ID NO.67.

[0155] In some preferred embodiments, the human CD3zeta intracellular signaling domain sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.68, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human CD3zeta intracellular signaling domain sequence is as shown in SEQ ID NO.68.

[0156] In some preferred embodiments, the human DAP10-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.69, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP10-CD3zeta sequence is as shown in SEQ ID NO.69.

[0157] In some preferred embodiments, the human DAP12-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.70, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP12-CD3zeta sequence is as shown in SEQ ID NO.70.

[0158] In some preferred embodiments, the human DAP10-DAP12-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.71, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP10-DAP12-CD3zeta sequence is as shown in SEQ ID NO.71.

[0159] In some preferred embodiments, the human 41BB intracellular signaling domain sequence comprises an amino acid sequence having 80% or greater identity to the amino acid sequence shown in SEQ ID NO. 72, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity; the amino acid sequence of the human 41BB intracellular signaling domain sequence is shown in SEQ ID NO. 72.

[0160] 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 an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or 99% or more identity; the amino acid sequence of the human DAP10-41BB sequence is as shown in SEQ ID NO.73.

[0161] In some preferred embodiments, the human DAP10-41BB-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.74, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP10-41BB-CD3zeta sequence is as shown in SEQ ID NO.74.

[0162] 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 an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or 99% or more identity; the amino acid sequence of the human CD28 intracellular signaling domain sequence is as shown in SEQ ID NO. 75.

[0163] In some preferred embodiments, the human DAP10-CD28 sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.76, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP10-CD28 sequence is as shown in SEQ ID NO.76.

[0164] In some preferred embodiments, the human DAP10-CD28-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.77, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP10-CD28-CD3zeta sequence is as shown in SEQ ID NO.77.

[0165] 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 an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or 99% or more identity; the amino acid sequence of the human DAP12-41BB sequence is as shown in SEQ ID NO.78.

[0166] In some preferred embodiments, the human DAP12-41BB-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.79, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP12-41BB-CD3zeta sequence is as shown in SEQ ID NO.79.

[0167] In some preferred embodiments, the human DAP12-CD28 sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.80, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP12-CD28 sequence is as shown in SEQ ID NO.80.

[0168] In some preferred embodiments, the human DAP12-CD28-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.81, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP12-CD28-CD3zeta sequence is as shown in SEQ ID NO.81.

[0169] In some embodiments, in the recombinant protein molecule that targets degradation of TCR in the UT component, the binding protein molecule domain targeting TCR or its functional variant is derived from a TCR antibody or its functional fragment or a combination thereof.

[0170] 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, double-chain antibody, three-chain antibody and four-chain antibody; preferably, the TCR antibody is a TCR single-chain antibody.

[0171] In some preferred embodiments, the ERAD degradation domain in the UT module is derived from HCMV glycoprotein US2, US3, US11 or US10, adenovirus E3-19K or HHV-7 US21.

[0172] In some preferred embodiments, the full-length sequence of the HCMV glycoprotein US2 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.82, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US2 is as shown in SEQ ID NO.82.

[0173] In some preferred embodiments, the HLA binding domain of the HCMV glycoprotein US2 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.83, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the HLA binding domain of the HCMV glycoprotein US2 is as shown in SEQ ID NO.83.

[0174] In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US2 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.84, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US2 is as shown in SEQ ID NO.84.

[0175] In some preferred embodiments, the full-length sequence of the HCMV glycoprotein US3 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.85, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US3 is as shown in SEQ ID NO.85.

[0176] In some preferred embodiments, the HLA binding domain of the HCMV glycoprotein US3 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.86, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the HLA binding domain of the HCMV glycoprotein US3 is as shown in SEQ ID NO.86.

[0177] In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US3 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.87, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US3 is as shown in SEQ ID NO.87.

[0178] In some preferred embodiments, the full-length sequence of the HCMV glycoprotein US11 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.88, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US11 is as shown in SEQ ID NO.88.

[0179] In some preferred embodiments, the MHC binding domain of the HCMV glycoprotein US11 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.89, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the MHC binding domain of the HCMV glycoprotein US11 is as shown in SEQ ID NO.89.

[0180] In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US11 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.90, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US11 is as shown in SEQ ID NO.90.

[0181] In some preferred embodiments, the full-length sequence of the HCMV glycoprotein US10 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.91, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US10 is as shown in SEQ ID NO.91.

[0182] In some preferred embodiments, the HLA binding domain of the HCMV glycoprotein US10 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.92, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the HLA binding domain of the HCMV glycoprotein US10 is as shown in SEQ ID NO.92.

[0183] In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US10 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.93, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US10 is as shown in SEQ ID NO.93.

[0184] In some preferred embodiments, the full-length sequence of the adenovirus E3-19K comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 94, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the full-length sequence of the adenovirus E3-19K is as shown in SEQ ID NO. 94.

[0185] In some preferred embodiments, the MHC binding domain of the adenovirus E3-19K comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 95, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the MHC binding domain of the adenovirus E3-19K is as shown in SEQ ID NO. 95.

[0186] In some preferred embodiments, the ERAD degradation domain of the adenovirus E3-19K comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.96, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the ERAD degradation domain of the adenovirus E3-19K is as shown in SEQ ID NO.96.

[0187] In some preferred embodiments, the full-length sequence of HHV-7US21 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.97, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the full-length sequence of HHV-7US21 is as shown in SEQ ID NO.97.

[0188] In some preferred embodiments, the MHC binding domain of HHV-7US21 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.98, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the MHC binding domain of HHV-7US21 is as shown in SEQ ID NO.98.

[0189] In some preferred embodiments, the ERAD degradation domain of HHV-7US21 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.99, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the ERAD degradation domain of HHV-7US21 is as shown in SEQ ID NO.99.

[0190] In some embodiments, the UT component further comprises (ii) a binding protein molecule domain or a functional variant thereof that targets MHC I and / or MHC II.

[0191] In some preferred embodiments, the binding protein molecule domain or its functional variant targeting MHC I and / or MHC II is a binding protein molecule domain or its functional variant targeting HLA.

[0192] In some preferred embodiments, the binding protein molecular domain targeting MHC I and / or MHC II or its functional variant is derived from a viral endoplasmic reticulum protein that inhibits the expression of MHC molecules or promotes their degradation; preferably, 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 its functional variant comprises a TAP binding domain.

[0193] In some preferred embodiments, the full-length sequence of HCMV US6 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.100, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of HCMV US6 is as shown in SEQ ID NO.100.

[0194] In some preferred embodiments, the TAP binding domain of HHV-7US6 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.101, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the TAP binding domain of HHV-7US6 is as shown in SEQ ID NO.101.

[0195] In some preferred embodiments, the full-length sequence of HSV ICP47 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.102, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of HSV ICP47 is as shown in SEQ ID NO.102.

[0196] In some preferred embodiments, the TAP binding domain of HSV ICP47 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.103, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the TAP binding domain of HSV ICP47 is as shown in SEQ ID NO.103.

[0197] In some preferred embodiments, the full-length sequence of CPXV012 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.104, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of CPXV012 is as shown in SEQ ID NO.104.

[0198] In some preferred embodiments, the TAP binding domain of CPXV012 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.105, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the TAP binding domain of CPXV012 is as shown in SEQ ID NO.105.

[0199] In some preferred embodiments, the full-length sequence of EBV BNFL2a comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.106, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the full-length sequence of EBV BNFL2a is as shown in SEQ ID NO.106.

[0200] In some preferred embodiments, the TAP binding domain of EBV BNFL2a comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.107, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the TAP binding domain of EBV BNFL2a is as shown in SEQ ID NO.107.

[0201] In some preferred embodiments, the full-length sequence of the BHV UL49.5 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.108, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the full-length sequence of the BHV UL49.5 is as shown in SEQ ID NO.108.

[0202] 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 an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or 99% or more identity; the amino acid sequence of the TAP binding domain of BHV UL49.5 is shown in SEQ ID NO.113.

[0203] In some preferred embodiments, the MHC I and / or MHC II binding protein molecule domain or its functional variant is derived from a viral glycoprotein that degrades MHC and / or MHC II molecules; preferably, the viral glycoprotein is selected from HCMV glycoprotein US2, US3, US11 or US10, adenovirus E3-19K or HHV-7US21.

[0204] In some preferred embodiments, the full-length sequence of US2 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.110, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of US2 is as shown in SEQ ID NO.110.

[0205] In some preferred embodiments, the HLA binding domain of US2 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.111, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the HLA binding domain of US2 is as shown in SEQ ID NO.111.

[0206] In some preferred embodiments, the ERAD degradation domain of US2 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.112, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the ERAD degradation domain of US2 is as shown in SEQ ID NO.112.

[0207] In some preferred embodiments, the full-length sequence of US3 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.113, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of US3 is as shown in SEQ ID NO.113.

[0208] In some preferred embodiments, the HLA binding domain of US3 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.114, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the HLA binding domain of US3 is as shown in SEQ ID NO.114.

[0209] In some preferred embodiments, the ERAD degradation domain of US3 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.115, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the ERAD degradation domain of US3 is as shown in SEQ ID NO.115.

[0210] In some preferred embodiments, the full-length sequence of US11 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.116, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of US11 is as shown in SEQ ID NO.116.

[0211] In some preferred embodiments, the HLA binding domain of US11 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.117, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the HLA binding domain of US11 is as shown in SEQ ID NO.117.

[0212] In some preferred embodiments, the ERAD degradation domain of US11 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.118, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the ERAD degradation domain of US11 is as shown in SEQ ID NO.118.

[0213] In some preferred embodiments, the binding protein molecular domain targeting MHC I and / or MHC II or its functional variant further comprises a viral protein that directs the inhibition or degradation of the NK target protein of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 or ULBP6; preferably, the viral protein is selected from HCMV UL16, UL141, UL142 or adenovirus E3-19K.

[0214] In some preferred embodiments, the full-length sequence of HCMV UL16 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 119, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of HCMV UL16 is as shown in SEQ ID NO. 119.

[0215] In some preferred embodiments, the NK target protein binding domain of HCMV UL16 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.120, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or 99% or more identity; the amino acid sequence of the NK target protein binding domain of HCMV UL16 is as shown in SEQ ID NO.120.

[0216] In some preferred embodiments, the ERAD degradation domain of HCMV UL16 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.121, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the ERAD degradation domain of HCMV UL16 is as shown in SEQ ID NO.121.

[0217] In some preferred embodiments, the full-length sequence of HCMV UL141 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 122, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of HCMV UL141 is as shown in SEQ ID NO. 122.

[0218] In some preferred embodiments, the NK target protein binding domain of HCMV UL141 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.123, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or 99% or more identity; the amino acid sequence of the NK target protein binding domain of HCMV UL141 is as shown in SEQ ID NO.123.

[0219] In some preferred embodiments, the ERAD degradation domain of HCMV UL141 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.124, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the ERAD degradation domain of HCMV UL141 is as shown in SEQ ID NO.124.

[0220] In some preferred embodiments, the full-length sequence of HCMV UL142 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 125, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of HCMV UL142 is as shown in SEQ ID NO. 125.

[0221] In some preferred embodiments, the MICA and ULBP3 binding domain of HCMV UL142 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 126, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or 99% or more identity; the amino acid sequence of the MICA and ULBP3 binding domain of HCMV UL142 is as shown in SEQ ID NO. 126.

[0222] In some preferred embodiments, the Golgi retention domain of HCMV UL142 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 127, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the Golgi retention domain of HCMV UL142 is as shown in SEQ ID NO. 127.

[0223] In some preferred embodiments, the binding protein molecule domain targeting MHC I and / or MHC II or its functional variant further comprises a viral protein that transports MHC I molecules from the Golgi apparatus to the lysosome for degradation; preferably, the viral protein is selected from HIV Nef, HIV Vpu, HHV-7U21, HHV-8KK3, HHV-8KK5, MHV-68MK3 and HTLV-1p12.

[0224] In some preferred embodiments, the HIV Nef comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.128, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the HIV Nef is as shown in SEQ ID NO.128.

[0225] In some preferred embodiments, the HIV Vpu comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.129, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the HIV Vpu is as shown in SEQ ID NO.129.

[0226] In some preferred embodiments, the HHV-8KK3 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.130, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the HHV-8KK3 is as shown in SEQ ID NO.130.

[0227] In some preferred embodiments, the HHV-8KK5 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.131, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the HHV-8KK5 is as shown in SEQ ID NO.131.

[0228] In some preferred embodiments, the MHV-68MK3 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.132, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the MHV-68MK3 is as shown in SEQ ID NO.132.

[0229] In some preferred embodiments, the HTLV-1p12 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO. 133, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the HTLV-1p12 is as shown in SEQ ID NO. 133.

[0230] In some preferred embodiments, the MHC I and / or MHC II-targeting binding protein molecular domain or its functional variant further comprises a viral protein that mediates the return of MHC-polypeptide molecules from the Golgi apparatus to the endoplasmic reticulum and promotes their degradation; preferably, the viral protein comprises an MHC binding structure and a KDEL receptor binding domain; preferably, the viral protein is the Cowpox Virus protein CPXV203.

[0231] In some preferred embodiments, the full-length sequence of the vaccinia virus protein CPXV203 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.134, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the full-length sequence of the vaccinia virus protein CPXV203 is as shown in SEQ ID NO.134.

[0232] In some preferred embodiments, the MHC binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO. 135, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the MHC binding domain of the vaccinia virus protein CPXV203 is as shown in SEQ ID NO. 135.

[0233] In some preferred embodiments, the KDEL receptor binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 136, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the KDEL receptor binding domain of the vaccinia virus protein CPXV203 is as shown in SEQ ID NO. 136.

[0234] In some preferred embodiments, the full-length sequence of the vaccinia virus protein CPXV203 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.137, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the full-length sequence of the vaccinia virus protein CPXV203 is as shown in SEQ ID NO.137.

[0235] In some preferred embodiments, the MHC binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO. 138, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the MHC binding domain of the vaccinia virus protein CPXV203 is as shown in SEQ ID NO. 138.

[0236] In some preferred embodiments, the KDEL receptor binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.139, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical; the amino acid sequence of the KDEL receptor binding domain of the vaccinia virus protein CPXV203 is as shown in SEQ ID NO.139.

[0237] In some embodiments, the multifunctional complex further comprises component (4) a chimeric adaptor component and / or a targeted tumor cell killing receptor component;

[0238] The (4) composite adapter comprises: (i) an extracellular recognition domain targeting a tumor; (ii) a transmembrane domain; and (iii) an intracellular signaling domain; optionally, a hinge or linker is included between the extracellular recognition domain targeting a tumor, the transmembrane domain and / or the intracellular signaling domain;

[0239] Preferably, the tumor-targeting extracellular recognition domain of the composite adapter component is selected from a tumor antigen-specific binding domain, a tumor microenvironment target antigen binding domain and / or a chemotactic receptor targeting the tumor microenvironment.

[0240] In some preferred embodiments, the tumor-targeting extracellular recognition domain is selected from an antibody or a functional fragment thereof, a TCR or a combination thereof that can target and recognize tumor-associated antigens; the functional fragment of the antibody is selected from Fd, Fv, Fab, Fab', F(ab')2, Fv (scFv), a single-chain antibody (scFv) or a nanobody, a double-chain antibody, a three-chain antibody and a four-chain antibody.

[0241] In some preferred embodiments, the transmembrane domain of the composite 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 their combination; preferably, 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; preferably, the natural cytotoxicity receptor is selected from NKp46, NKp44 and NKp30.

[0242] In some preferred embodiments, the intracellular signaling domain of the composite adapter component includes the intracellular signaling domain and / or the co-stimulatory signaling domain of an NK cell activating receptor.

[0243] In some preferred embodiments, 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.

[0244] In some preferred embodiments, the intracellular signaling domain further comprises a co-stimulatory signaling domain; preferably, the co-stimulatory signaling domain is selected from T cell co-stimulatory signaling domains; including, but not limited to, those 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, 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, VLA 1. CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITG B1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, NCR, DAP10, DAP12, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), C D84, 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 ligands that specifically bind to CD83, intracellular signaling domains such as CARD11, FcRa, FcRp, FcRy, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, Wnt, OX40, ROR2, Ryk, SLAMF1, Slp76, pTa, TCRa, TCRp, TRIM, ZAP70, and PTCH2;More preferably, the costimulatory signaling domain is selected from the group consisting of an NKG2D intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, an NCR intracellular signaling domain, a CD28 intracellular signaling domain, a 4-1BB intracellular signaling domain, an OX40 intracellular signaling domain, and an ICOS intracellular signaling domain;

[0245] The targeted tumor cell killing receptor component comprises (i) an extracellular recognition domain targeting a tumor antigen; (ii) a transmembrane domain; and (iii) an intracellular co-stimulatory signaling domain; (iv) a T cell activation signaling domain (ITAM); optionally, a hinge or linker is contained between the extracellular recognition domain targeting a tumor antigen, the transmembrane domain, the intracellular co-stimulatory signaling domain and / or the T cell activation signaling domain (ITAM);

[0246] The transmembrane domain of the targeted tumor cell receptor component is selected from the group consisting of a CD8 transmembrane domain, an α and / or β chain transmembrane domain of a T cell receptor, a CD28 transmembrane domain, a CD3ε transmembrane domain, a CD45 transmembrane domain, a CD4 transmembrane domain, a CD5 transmembrane domain, a CD8 transmembrane domain, a CD9 transmembrane domain, a CD16 transmembrane domain, a CD22 transmembrane domain, a CD33 transmembrane domain, a CD37 transmembrane domain, a CD64 transmembrane domain, a CD80 transmembrane domain, a CD86 transmembrane domain, a CD134 transmembrane domain, a CD137 transmembrane domain, a CD154 transmembrane domain, a GITR transmembrane domain, and combinations thereof;

[0247] The T cell activation signaling domain is derived from intracellular signaling domains such as CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI CD66d, DAP10 and DAP12.

[0248] In some preferred embodiments, the linker is a flexible linker; preferably, the flexible linker comprises the amino acid sequence shown (Gly(x)Ser(y))n, wherein n is an integer from 1 to 10, and x and y are independently integers from 0 to 10, with the proviso that x and y are not both 0; more preferably, the linker comprises the amino acid sequence shown in SEQ ID NO.140 or the amino acid sequence shown in SEQ ID NO.141.

[0249] In some preferred embodiments, the linker is a hinge; preferably, the hinge is an IgG1 hinge or an IgG4 hinge.

[0250] In some preferred embodiments, the IgG1 hinge comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.142, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the IgG1 hinge is as shown in SEQ ID NO.142.

[0251] In some preferred embodiments, the IgG4 hinge comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.143, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the IgG4 hinge is as shown in SEQ ID NO.143.

[0252] In some preferred embodiments, a cleavable peptide is contained between the NK activating receptor component, CNK signaling transduction component and / or UT component; for example, a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, a F2A peptide, a GSG-F2A peptide, a P2A peptide or a GSG-P2A peptide.

[0253] In some preferred embodiments, the T2A comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.144, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of T2A is as shown in SEQ ID NO.144.

[0254] In some preferred embodiments, the amino acid sequence of the GSG-T2A peptide is shown as SEQ ID NO.145.

[0255] In some preferred embodiments, the P2A comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.146, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the P2A is as shown in SEQ ID NO.146.

[0256] In some preferred embodiments, the amino acid sequence of the GSG-P2A peptide is shown as SEQ ID NO.147.

[0257] In some preferred embodiments, the E2A comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 148, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the E2A is as shown in SEQ ID NO. 148.

[0258] In some preferred embodiments, the amino acid sequence of the GSG-E2A peptide is shown as SEQ ID NO.149.

[0259] In some preferred embodiments, the F2A comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 150, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or 99% or more identity; the amino acid sequence of F2A is shown in SEQ ID NO. 150.

[0260] In some preferred embodiments, the amino acid sequence of the GSG-F2A peptide is shown as SEQ ID NO.151.

[0261] In some preferred embodiments, the multifunctional complex comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.152, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the multifunctional complex comprises a TCR antibody single-chain antibody as shown in SEQ ID NO.152.

[0262] In some preferred embodiments, the multifunctional complex comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.153, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the multifunctional complex is as shown in SEQ ID NO.153.

[0263] In one aspect, the present disclosure provides a nucleic acid molecule encoding the multifunctional complex.

[0264] In some preferred embodiments, the nucleic acid molecule is DNA or RNA.

[0265] In some preferred embodiments, the RNA is mRNA.

[0266] In some preferred embodiments, the nucleic acid molecule comprises a nucleotide sequence that is 80% or more identical to the nucleotide sequence shown in SEQ ID NO. 154, preferably a nucleotide sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical.

[0267] In one aspect, the present disclosure provides an expression vector containing the nucleic acid.

[0268] In some preferred embodiments, the vector is selected from a plasmid, a cosmid, a viral vector, an RNA vector, or a linear or circular DNA or RNA molecule.

[0269] In some preferred embodiments, the viral vector is selected from a retrovirus, an adenovirus, a parvovirus (e.g., an adeno-associated virus), a coronavirus, a negative-strand RNA virus such as an orthomyxovirus (e.g., influenza virus), a rhabdovirus (e.g., rabies and vesicular stomatitis virus), a paramyxovirus (e.g., malabsorption and Sendai), a positive-strand RNA virus such as a picornavirus and an alphavirus, and a double-stranded DNA virus, including adenovirus, 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), a Norwalk virus, a togavirus, a flavivirus, a reovirus, a papovavirus, a hepadnavirus, a baculovirus and a hepatitis virus.

[0270] In some preferred embodiments, the retrovirus is selected from the group consisting of avian leukocytoblastoma, mammalian C-type, B-type, D-type virus, HTLV-BLV collection, lentivirus, and foamy virus.

[0271] In some preferred embodiments, the lentiviral vector is selected from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or ovine demyelinating leukoencephalitis lentivirus.

[0272] 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 under the same promoter, or under different promoters, or can be expressed in multiple vectors.

[0273] In some preferred embodiments, the vector is a lentiviral vector, and a cleavable peptide encoding gene is contained between the genes encoding the NK activation receptor component, the CNK signal transduction component and / or the UT component; preferably, the cleavable peptide is a 2A linker; and the 2A linker is selected from T2A, P2A, E2A and F2A.

[0274] In some preferred embodiments, the vector further comprises a promoter; preferably, the promoter is an EF1α promoter or a CMV promoter.

[0275] In one aspect, the present disclosure provides an immune cell comprising the nucleic acid or the expression vector.

[0276] In some preferred embodiments, the immune cells are selected from T cells, NKT cells, NK cells, B cells, monocytes, macrophages, etc.

[0277] In one aspect, the present disclosure provides a method for producing immune cells, comprising introducing the nucleic acid or the expression vector into cells using a method selected from the following: electroporation, sonoporation, a gene gun (e.g., a gene gun using Au-particles), lipofection, polymer transfection, nanoparticles or multiplexes.

[0278] In one aspect, the present disclosure provides a pharmaceutical composition comprising the multifunctional complex, the nucleic acid, the expression vector, the immune cell and / or the immune cell produced by the method, and a pharmaceutically acceptable carrier.

[0279] In one aspect, the present disclosure provides a use of 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 preparing a drug for treating a disease.

[0280] In one aspect, the present disclosure provides a method for treating a disease, comprising administering the multifunctional complex, the nucleic acid, the expression vector, the immune cell and / or the pharmaceutical composition to a subject.

[0281] In some preferred embodiments, the diseases include various solid tumors and blood tumors, viral infectious diseases, and autoimmune diseases.

[0282] In some preferred embodiments, the solid tumor is selected from the group consisting of nervous system tumors, head and neck tumors, chest tumors, digestive system tumors, genitourinary system tumors, soft tissue and skin tumors, bone tumors, and the like.

[0283] In some preferred embodiments, nervous system tumors include diffuse gliomas, diffuse astrocytomas and anaplastic astrocytomas, glioblastomas, oligodendrogliomas, oligoastrocytomas, childhood diffuse gliomas, other astrocytomas, ependymomas, neuronal and mixed neuronal-glial tumors, medulloblastomas, other embryonal tumors, schwannomas, meningiomas, solitary fibrous tumors and hemangiopericytomas, etc.

[0284] In some preferred embodiments, head and neck tumors include nasal cavity and paranasal sinus malignancies, nasopharyngeal cancer, oral cancer, laryngeal cancer, salivary gland tumors, intracranial tumors, thyroid cancer, tongue cancer, etc.

[0285] In some preferred embodiments, the thoracic tumors include lung cancer, esophageal cancer, cardiac cancer, breast cancer, mediastinal tumors, and the like.

[0286] In some preferred embodiments, digestive system tumors include gastric cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, small intestinal malignancies, etc.

[0287] In some preferred embodiments, the genitourinary system tumors include renal cancer, prostate cancer, bladder cancer, testicular malignancies, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, and the like.

[0288] In some preferred embodiments, soft tissue and skin tumors include malignant fibrous histiocytoma, rhabdomyosarcoma, synovial sarcoma, malignant melanoma of the skin, and the like.

[0289] In some preferred embodiments, bone tumors include osteosarcoma, Ewing's sarcoma, and the like.

[0290] In some preferred embodiments, the colon cancer is a colon adenoma.

[0291] In some preferred embodiments, the breast cancer is triple-negative breast cancer cells.

[0292] In some preferred embodiments, the liver cancer is hepatocellular carcinoma.

[0293] In some preferred embodiments, the disease is a blood tumor selected from leukemia, lymphoma (HL), multiple myeloma (MM), myelodysplastic syndrome (MDS), etc.

[0294] In some preferred embodiments, the leukemia is B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute myeloid leukemia, etc.

[0295] In some preferred embodiments, viral infectious diseases include: respiratory viral diseases, gastrointestinal viral diseases, liver viral diseases, skin and mucous membrane viral diseases, eye viral diseases, central nervous system viral diseases, lymphocytic viral diseases, insect-borne viral diseases, slow virus infectious diseases, etc.

[0296] In some preferred embodiments, respiratory viral diseases include infections caused by rhinovirus, adenovirus, respiratory syncytial virus, parainfluenza virus, and coronavirus; influenza; mumps; and the like.

[0297] In some preferred embodiments, gastrointestinal viral diseases include poliomyelitis; Kuksaki virus infection; ECHO virus infection; viral gastroenteritis: including rotavirus gastroenteritis, Norwalk virus gastroenteritis, adenovirus gastroenteritis, astrovirus gastroenteritis, coronavirus gastroenteritis and calicivirus gastroenteritis, etc.

[0298] In some preferred embodiments, viral liver diseases include hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, Epstein-Barr virus hepatitis, cytomegalovirus hepatitis, and the like.

[0299] In some preferred embodiments, viral diseases of the skin and mucous membranes include measles, rubella, roseola infantum, varicella and herpes zoster, smallpox, herpes simplex virus infection, rabies and foot-and-mouth disease.

[0300] In some preferred embodiments, the ocular viral diseases include epidemic keratoconjunctivitis, follicular conjunctivitis, and herpes keratoconjunctivitis.

[0301] In some preferred embodiments, viral diseases of the central nervous system include Japanese encephalitis, Western equine encephalitis, Eastern equine encephalitis, St. Louis encephalitis, Venezuelan equine encephalitis, Murray Valley encephalitis, California encephalitis, forest encephalitis, and lymphocytic choriomeningitis.

[0302] In some preferred embodiments, the lymphocytic viral diseases include infectious mononucleosis, cytomegalovirus infection, and acquired immunodeficiency syndrome.

[0303] In some preferred embodiments, the insect-borne viral diseases include viral hemorrhagic fevers, including epidemic hemorrhagic fever, yellow fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever, Omsk hemorrhagic fever, Marburg disease, and Ebola hemorrhagic fever; dengue fever and dengue hemorrhagic fever; West Nile fever; Colorado tick fever; sandfly fever;

[0304] Preferably, the lentiviral infectious diseases include subacute sclerosing panencephalitis, kuru, progressive multifocal leukoencephalopathy and subacute spongiform encephalopathy (corticostriatal degeneration), etc.

[0305] In some preferred embodiments, the autoimmune disease includes organ-specific autoimmune diseases and systemic autoimmune diseases;

[0306] Preferably, the organ-specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, etc.

[0307] In some preferred embodiments, the systemic autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, etc.

[0308] In one aspect, the present disclosure provides a method for stimulating an immune response in a subject, comprising administering to the subject an effective amount of the multifunctional complex, the nucleic acid, the expression vector, the immune cell, the immune cell produced by the method, and / or the pharmaceutical composition.

[0309] The present 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 therapeutic methods, for administering the cells and compositions to a subject, such as a patient.

[0310] 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.

[0311] In one embodiment, the present invention provides a chimeric protein construct for targeting a specific protein (i.e., a target protein) to block its expression (e.g., surface expression) and accelerate ubiquitination-mediated degradation through the endoplasmic reticulum-associated degradation (ER-associated degradation, ERAD) mechanism.

[0312] Endoplasmic reticulum-associated protein degradation (ERAD) refers to a cellular pathway that targets misfolded proteins in the endoplasmic reticulum for ubiquitination and subsequent degradation by the proteasome. The ERAD process can be divided into three steps: (1) 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: terminally misfolded proteins must be transported from the endoplasmic reticulum back to the cytoplasm, which contains the ubiquitin-proteasome system (UPS); (3) ubiquitin-dependent degradation of the identified misfolded or mutated proteins by the proteasome (see Annamaria et al., ER-associated degradation: Protein quality control and beyond. J. Cell Biol. Vol. 204 No. 6 869–879).

[0313] Here, the present application provides a new chimeric strategy that fuses a targeted protein binding domain (such as a specific protein targeting antibody fragment (ScFv)) with an ERAD functional motif (such as a viral ER resident protein TMD and ED return signal domain), and demonstrates that this strategy can effectively inhibit the expression of a specific protein (for example, the expression of a specific protein on the cell surface).

[0314] The TPD technology platform provided in this application has the following advantages over existing protein degradation / removal technologies (e.g., PROTAC, CRISPR, etc.): 1. The ER-TPD chimeric protein construct is delivered to the cell through a carrier (e.g., liposomes, nanoparticles, lentivirus, adenovirus, oncolytic virus, etc.) for stable expression and function. Compared with PROTAC, there is no problem of low cell permeability and short half-life leading to low degradation efficiency; 2. ER-TPD technology is based on the ERAD principle of the endoplasmic reticulum, which directly intervenes in protein synthesis and achieves efficient degradation during the synthesis and assembly of endoplasmic reticulum proteins. It can achieve degradation of cell membrane surface proteins and secretory proteins at the source, which are proteins that PROTAC cannot target. Therefore, ER-TPD can target a large number of proteins that conventional technologies cannot target and degrade; 3. In the ER-TPD design method, the targeting domain can adopt various single-chain antibody structures or artificial affinity ligand structures to greatly realize the specific recognition and binding ability of the target protein; while conventional PROTAC technology and conventional PROTAC 4. ER-TPD technology uses a small molecule chemical structure, which makes it difficult to ensure the specificity of the targeting structure. Therefore, ER-TPD technology has the strong advantages of targeting accuracy and specificity. 5. ER-TPD technology can achieve a multi-pathway mechanism for target protein degradation through downstream degradation-guided ligand structures, including the UPS mechanism based on the ubiquitin proteasome and the ALP mechanism of the autophagy lysosome. The downstream degradation-guided ligand structure design can flexibly connect the degradation system in the cell through genetic engineering methods to achieve efficient targeted degradation, while conventional PROTAC technology can only degrade by targeting E3 ligases. 6. 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 are no toxic side effects such as chromosomal instability caused by off-target effects or gene editing processes. 7. The development of targeted degradation drugs based on ER-TPD technology is highly feasible and has a short cycle. At the same time, the production cost of nucleic acids and cell drugs based on ER-TPD technology is low, and it is easier to achieve scale and industrialization.

[0315] In one aspect, the present disclosure provides a chimeric protein construct of endoplasmic reticulum-based targeted protein degradation technology (ER-TPD), which comprises an endoplasmic reticulum-associated degradation (ERAD) machinery protein binding domain and a targeting protein binding domain.

[0316] In certain embodiments, the chimeric protein construct further comprises a protein degradation pathway member (e.g., a ubiquitin-proteasome system pathway member, an endosome-lysosome pathway member, an autophagy pathway member) binding domain. The protein degradation pathway member binding domain can be linked to the constituent elements of the chimeric protein construct in any suitable manner, for example, to the ERAD machinery protein binding domain.

[0317] In another aspect, the present disclosure also provides a library of compositions, wherein each composition comprises a viral protein or a nucleic acid molecule encoding the same, which comprises a functional motif capable of hijacking the ERAD machinery to arrest and accelerate proteasome-mediated degradation within the ER and an affinity portion that targets a specific protein for degradation, for therapeutic purposes to modify cell phenotype, improve cell function, induce apoptosis, or inhibit viral replication.

[0318] 1. ERAD mechanism protein binding domain

[0319] As used herein, the term "ERAD machinery protein" refers to a protein that participates in the ERAD mechanism or pathway. The term "ERAD machinery protein binding domain", also referred to as "ERAD degradation domain", refers to a portion or domain that can bind to and / or utilize an ERAD machinery protein, for example, a transmembrane domain (or a functional variant thereof) and a cytoplasmic domain (or a functional variant thereof) of a viral endoplasmic reticulum (ER) resident glycoprotein, the cytoplasmic domain also referred to as an "endoplasmic reticulum retention domain" or "ER retention domain".

[0320] In some embodiments, the ERAD machinery protein binding domain comprises a transmembrane domain of a viral endoplasmic reticulum resident protein or a functional variant thereof and an endoplasmic reticulum resident domain or a functional variant thereof. In some embodiments, the viral ER resident glycoprotein can be any viral ER resident protein that is capable of hijacking the ERAD machinery and promoting ubiquitination and proteasome-mediated degradation of target proteins.

[0321] i. ERAD machinery protein binding domain derived from adenovirus E3-19K

[0322] In some embodiments, the viral endoplasmic reticulum-resident protein is adenovirus E3-19K. Adenovirus E3-19K (also known as "E19") contains three functional modules: a lumenal domain for interacting with MHC-I and MICA / B molecules, a transmembrane domain, and an endoplasmic reticulum-resident domain, wherein its endoplasmic reticulum-resident domain contains a dilysine motif in the cytoplasmic tail that can return the Golgi apparatus to the endoplasmic reticulum, and therefore can also be called an "ER return signal motif". Studies have shown that the transmembrane domain and the ER return signal motif are required to ensure effective ER localization, transport inhibition of the histocompatibility complex class I (MHC-I) and MHC-I-related chains A and B (MICA / B molecules), and proteasomal degradation. Therefore, adenovirus E3-19K can reside MHC class I molecules (e.g., MHC-I and MICA / B molecules) in the secretory pathway and interfere with antigen presentation. The chimeric protein construct provided by the present disclosure utilizes the transmembrane domain and endoplasmic reticulum retention domain (ie, ERAD degradation domain) of proviral E3-19K to fuse with the targeting protein binding domain, thereby achieving the purpose of target protein degradation.

[0323] In some preferred embodiments, the ERAD degradation domain of the adenovirus E3-19K comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 96, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity. The ERAD degradation domain of the adenovirus E3-19K has a dilysine motif in the cytoplasmic tail, which is capable of returning the Golgi apparatus to the endoplasmic reticulum. In some preferred embodiments, the amino acid sequence of the ERAD degradation domain of the adenovirus E3-19K is as shown in SEQ ID NO. 96.

[0324] ii. ERAD machinery protein binding domains derived from other viral glycoproteins

[0325] In some embodiments, the viral ER-resident protein is not adenoviral E3-19K.

[0326] 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 glycoprotein US2 and US11.

[0327] 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.

[0328] a) HCMV US2 and US11

[0329] Two HCMV proteins, US2 and US11, are ER-resident type I integral membrane glycoproteins that co-opt the ERAD pathway to promote the degradation of MHC class I heavy chains, thereby inhibiting MHC class I antigen presentation. Expression of either protein results in rapid degradation of newly synthesized MHC class I heavy chains. US2 and US11 bind to MHC class I heavy chains through their luminal domains and recruit host cell proteins that extract the polypeptides from the ER membrane by "pulling" on the cytoplasmic tail of the heavy chain. Following translocation into the cytoplasm, MHC class I molecules are ubiquitinated and degraded by the proteasome.

[0330] In addition to class I molecules, US2 also leads to the degradation of two proteins of the class II pathway, DR-α and DM-α, as well as HFE, a nonclassical major histocompatibility complex (MHC) class I protein involved in iron regulation.

[0331] The lumenal domain of US2 is responsible for binding to MHC class I and class II molecules, and the transmembrane domain (TM) and cytoplasmic domain (CT) interact with cellular components of the ERAD mechanism or pathway and contribute to translocation and promote the enzymatic degradation of both MHC class I and class II proteins (Chevalier MS et al., 2002, 2003). The cytoplasmic tail of US2 is sufficient to interact with signal peptide peptidase (SPP), which is an essential component of the US2-dependent MHC I dislocation complex (Loureiro J et al., 2006) 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 ER-resident RING-type E3 ligase TRC8 through its TM domain, which also facilitates the ubiquitination and proteasomal degradation of US2-tail-anchored MHC I and II molecules (Stagg HR et al., 2009).

[0332] In contrast, US11-induced degradation of MHC-I molecules requires Derlin-1, but not SPP. The ER luminal domain of US11 interacts with the luminal domain of MHC-I heavy chains, while the TM domain of US11 binds to Derlin-1. Therefore, the primary function of US11 may be to deliver MHC-I molecules to Derlin-1 (Lilley BN et al., 2004; Cho S et al., 2013), which then induces their translocation to the cytosol for proteasomal degradation. Furthermore, US11 activates unfolded proteins. Through Derlin-1, US11 associates with TMEM129, an E3 ligase in the ERAD ring, and recruits Ube2J2 to ubiquitinate MHC-I prior to US11-induced degradation.

[0333] In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US2 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 84, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has the function of endoplasmic reticulum retention. In some embodiments, the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US2 is as shown in SEQ ID NO. 84.

[0334] In some preferred embodiments, the ERAD degradation domain of US2 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence shown in SEQ ID NO. 112, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has the function of endoplasmic reticulum retention. In some embodiments, the amino acid sequence of the ERAD degradation domain of US2 is as shown in SEQ ID NO. 112.

[0335] In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US11 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 90, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has the function of endoplasmic reticulum retention. In some embodiments, the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US11 is as shown in SEQ ID NO. 90.

[0336] In some preferred embodiments, the ERAD degradation domain of US11 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence shown in SEQ ID NO. 118, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has the function of endoplasmic reticulum retention. In some embodiments, the amino acid sequence of the ERAD degradation domain of US11 is as shown in SEQ ID NO. 118.

[0337] b) HCMV US3

[0338] Rather than promoting degradation of MHC proteins, the HCMV US3 glycoprotein physically associates with peptide-loaded MHC class I heterodimers, resulting in retention of the class I complex in the ER and inhibiting binding of the invariant chain to class II DR-αβ dimers in the ER, leading to mislocalization of the class II complex and reduced peptide loading. Thus, during the early stages of HCMV infection, US3 interferes with the intracellular trafficking and maturation of MHC class I molecules. US3 is an ER-resident membrane protein. Domain swapping experiments have demonstrated that the lumenal domain of US3 is sufficient for US3 itself to remain in the ER, while both the lumenal domain and the transmembrane domain are required for ER retention of MHC class I molecules.

[0339] In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US3 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 87, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has the function of endoplasmic reticulum retention. In some embodiments, the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US3 is as shown in SEQ ID NO. 87.

[0340] In some preferred embodiments, the ERAD degradation domain of US3 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence shown in SEQ ID NO. 115, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has the function of endoplasmic reticulum retention. In some embodiments, the amino acid sequence of the ERAD degradation domain of US3 is as shown in SEQ ID NO. 115.

[0341] c) HCMV US10

[0342] The HCMV US10 glycoprotein also interacts with components of the MHC class I antigen presentation complex. US10 binds to free class I heavy chains and delays their trafficking from the ER. However, US10 does not affect US2 or US11.

[0343] In some preferred embodiments, the ERAD degradation domain of the HCMV glycoprotein US10 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 93, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the ERAD degradation domain of the HCMV glycoprotein US10 is as shown in SEQ ID NO. 93.

[0344] d)HCMV US6, HSV ICP47, CPXV012, EBV BNFL2a, BHV UL49.5

[0345] Unlike US2, US3, US10 and US11, the HCMV L protein US6 affects antigen presentation through a completely different strategy. Instead of interacting with free class I heavy chains or fully assembled class I complexes, US6 inhibits the translocation of cytoplasmic peptides through the TAP complex (TAP1 / 2). US6 binds to the ER luminal side of TAP1 and causes a conformational change, thereby preventing the binding of ATP. Residues 89-108 in the ER-luminal domain of US6 contribute to the binding of US6 to TAP and are necessary and sufficient for this inhibition. This inhibition of TAP activity not only affects the expression of classical MHC class I alleles, but also affects the expression of non-classical alleles HLA-C and HLA-G in fetal cytotrophoblast cells.

[0346] HSV ICP47, a HCMV US6 protein, is expressed early in the infectious cycle and is dispensable for in vitro replication. Similar strategies can be applied to block class I molecule assembly. ICP47 blocks TAP-mediated peptide transport and binds tightly to the TAP1-TAP2 complex. A clue to the mechanism by which ICP47 blocks TAP is its high species selectivity. Both HSV1 and HSV2 ICP47 inhibit simian, monkey, porcine, dog, and bovine TAP, with little effect on mouse, rat, guinea pig, or rabbit TAP. The affinity of ICP47 for human TAP is approximately 100-fold higher than for mouse TAP. ICP47 inhibits peptide binding to TAP but does not affect ATP binding. With an affinity for TAP 10- to 1000-fold higher than that of most peptides, ICP47 acts as a competitive inhibitor of peptide binding to TAP and is thought to bind directly to the peptide binding site.

[0347] In some preferred embodiments, the TAP binding domain of HHV-7US6 comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO. 101, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity, and more preferably an amino acid sequence having 98% or 99% or more identity, and has the function of endoplasmic reticulum retention. In some embodiments, the amino acid sequence of the TAP binding domain of HHV-7US6 is as shown in SEQ ID NO. 101.

[0348] In some preferred embodiments, the TAP binding domain of HSV ICP47 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence shown in SEQ ID NO. 103, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has the function of endoplasmic reticulum retention. In some embodiments, the amino acid sequence of the TAP binding domain of HSV ICP47 is as shown in SEQ ID NO. 103.

[0349] In some preferred embodiments, the TAP-binding domain of CPXV012 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 105, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the TAP-binding domain of CPXV012 is as shown in SEQ ID NO. 105.

[0350] In some preferred embodiments, the TAP-binding domain of EBV BNFL2a comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 107, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the TAP-binding domain of EBV BNFL2a is as shown in SEQ ID NO. 107.

[0351] In some preferred embodiments, the TAP-binding domain of BHV UL49.5 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 109, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the TAP-binding domain of BHV UL49.5 is as shown in SEQ ID NO. 109.

[0352] e) Others

[0353] In some preferred embodiments, the ERAD degradation domain of HHV-7US21 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 99, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the ERAD degradation domain of HHV-7US21 is as shown in SEQ ID NO. 99.

[0354] In some preferred embodiments, the ERAD degradation domain of HCMV UL16 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 121, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the ERAD degradation domain of HCMV UL16 is as shown in SEQ ID NO. 121.

[0355] In some preferred embodiments, the ERAD degradation domain of HCMV UL141 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 124, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the ERAD degradation domain of HCMV UL141 is as shown in SEQ ID NO. 124.

[0356] In some preferred embodiments, the Golgi retention domain of HCMV UL142 comprises an amino acid sequence that is 80% or greater identical to the amino acid sequence of SEQ ID NO. 127, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identical, and more preferably an amino acid sequence that is 98% or greater identical, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the Golgi retention domain of HCMV UL142 is as shown in SEQ ID NO. 127.

[0357] In some preferred embodiments, the HIV Nef comprises an amino acid sequence that is 80% or greater identical to the amino acid sequence of SEQ ID NO. 128, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identical, and more preferably an amino acid sequence that is 98% or greater identical, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the HIV Nef is as shown in SEQ ID NO. 128.

[0358] In some preferred embodiments, the HIV Vpu comprises an amino acid sequence that is 80% or greater identical to the amino acid sequence of SEQ ID NO. 129, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identical, and more preferably an amino acid sequence that is 98% or greater identical, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the HIV Vpu is as shown in SEQ ID NO. 129.

[0359] In some preferred embodiments, the HHV-8KK3 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence shown in SEQ ID NO. 130, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the HHV-8KK3 is as shown in SEQ ID NO. 130.

[0360] In some preferred embodiments, the HHV-8KK5 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 131, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the HHV-8KK5 is as shown in SEQ ID NO. 131.

[0361] In some preferred embodiments, the MHV-68MK3 comprises an amino acid sequence that is 80% or greater identical to the amino acid sequence of SEQ ID NO. 132, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identical, and more preferably an amino acid sequence that is 98% or greater identical, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the MHV-68MK3 is as shown in SEQ ID NO. 132.

[0362] In some preferred embodiments, the HTLV-1p12 comprises an amino acid sequence that is 80% or greater identical to the amino acid sequence of SEQ ID NO. 133, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identical, and more preferably an amino acid sequence that is 98% or greater identical, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the HTLV-1p12 is as set forth in SEQ ID NO. 133.

[0363] In some preferred embodiments, the KDEL receptor-binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 136, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the KDEL receptor-binding domain of the vaccinia virus protein CPXV203 is as shown in SEQ ID NO. 136.

[0364] In some preferred embodiments, the KDEL receptor-binding domain of the vaccinia virus protein CPXV203 comprises an amino acid sequence having 80% or greater identity to the amino acid sequence of SEQ ID NO. 139, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity, and has endoplasmic reticulum retention function. In some embodiments, the amino acid sequence of the KDEL receptor-binding domain of the vaccinia virus protein CPXV203 is as shown in SEQ ID NO. 139.

[0365] 2. Targeting domain

[0366] The target binding domain of the chimeric protein constructs provided herein can be any structure that recognizes and binds to a target protein, such as an antibody, an antibody fragment, a functional motif derived from a natural protein (e.g., a natural ligand), an artificially synthesized polypeptide or protein that has affinity for the target protein, or various variants of the aforementioned molecules, such as mutants, fusions, and truncations. For example, the targeting domain can be a natural ligand of the target protein, an antibody or antigen-binding fragment thereof that specifically recognizes the target protein, or an antigen that can be specifically recognized by an antibody.

[0367] In some embodiments, the targeting domain comprises an antibody or a functional fragment thereof that specifically targets a target protein (e.g., a diabody, Fab, Fab', F(ab')2, Fd, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (dsdiabody), a single-chain antibody molecule (scFv), a scFv dimer (bivalent diabody), Fv (scFv), a multispecific antibody (e.g., a bispecific antibody), a camelized single domain antibody (e.g., VHH), a nanobody, a domain antibody, a bivalent domain antibody, a diabody, a triabody, and a tetrabody). The targeting protein domain may also comprise any binding ligand capable of binding to a target protein, including but not limited to a ligand, a portion of a receptor that binds to a ligand, a portion of a viral protein that binds to a host protein, and the like.

[0368] The target protein can be any protein whose level or activity is desired to be modulated, for example, by altering its activity through the ERAD mechanism to block proteins in the ER and accelerate proteasome-mediated degradation, the degradation of which can change the phenotype of the cell, improve cell function, induce cell apoptosis and / or inhibit viral replication, etc., thereby achieving therapeutic purposes (e.g., improving efficacy, reducing side effects, etc.). In some embodiments, the target protein can be a protein involved in the cell cycle, apoptosis, signal transduction, cell differentiation, cell dedifferentiation, cell growth, the production of cytokines or their biological regulators, the production of cytokines or biological regulators, the production of regulatory or functional proteins, pro-inflammatory signaling, or glucose regulation pathways.

[0369] In another embodiment, the target protein is a disease-associated protein (also referred to as a "pathogenic protein"). The "disease-associated protein" or "pathogenic protein" described herein refers to any protein whose function or activity changes will lead to the occurrence of the disease, or whose function is important for the development of the disease. Exemplary pathogenic proteins include but are not limited to oncoproteins, viral proteins, and autoimmune disease-causing proteins (e.g., antibodies produced by plasma cells).

[0370] In some embodiments, the target protein is selected from: target proteins related to immune function, target proteins related to nervous system diseases, target proteins related to infectious diseases (such as target proteins related to viral infections), target proteins related to self-antigens or target proteins related to tumors (or oncogenic proteins), and target proteins related to metabolic diseases.

[0371] In some embodiments, the pathogenic protein is an oncoprotein. The oncoprotein may be encoded by an oncogene, including but not limited to BCL2, c-MYC, Ras, HER2, BCR / ABL, ABL1 / BCR, TGFB1, TLX1, P53, WNT1, WNT2, WT1, αv-β3, PKCa, ABL, BCL1, CD24, CDK4, EGFR / ERBB-1, HSTF1, INT1 / WNT1, INT2, MDM2, MET, MYB, MYC, MYCN, MYCL1, RAFI, NRAS, REL, AKT2, APC, BCL2-ALPHA, BCL2-BETA, BCL3, BCR, BRCA1, BRCA 2. 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, FLI 1 / 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, KR AS2B, 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, T IAM1, 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-CA D. 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,In some embodiments, the oncoprotein may be a member of the Bcl-2 family (e.g., Bcl-2, Bcl-xL, and Bcl-xL). Bcl-w), VEGF / VEGFR, PDGFRβ, EGFR, EGFR mutant, IGF-1R, HDACs, HER2, MYC, KRAS, AFP, CEA, CA199, estrogen receptor ER-α, androgen receptor receptor, AR), tyrosine kinases (c-ABL, BCR-ABL, BTK, FAK, PTK6, Wee1, TRK transmembrane receptors), serine / threonine kinase receptors (IRAK4, LRRK2, B-Raf, RIPK2, CDK4 / 6, CDK7, CDK8, CDK8 / 19, CDK9, TBK1), protein kinase II (CK2), epigenetic-related proteins (BRD2, BRD3, BRD4, BRDT, TRIM24, BRD9, PBRM1, SMARCA2, SMARCA4, EP300, EZH2, WDR5), adrenomedullin (ADM), DPP3.

[0372] In some embodiments, the target protein includes immune function-related proteins. As used herein, the term "immune function-related proteins" refers to functional proteins involved in the body's immune process, such as antigen presenting molecules (e.g., MHC class I molecules, MHC class II molecules, HLA, etc.), antigen recognition molecules (e.g., TCR, CD123, NKG2D, etc.), immune checkpoint molecules (e.g., PD-1, PD-L1, CTLA4, TIM3, TIGIT, LAG3, A2AR, BTLA, IDO1, IDO2, TDO, KIR, NOX2, VISTA, SIGLEC7, PVR, etc.), immunostimulatory / co-stimulatory molecules (e.g., CD3, CD80 / 86, CD28, etc.), and other molecules involved in innate or adaptive immunity. In some embodiments, target proteins associated with immune function include, for example, CD123, CD7, CD5, MHC class I molecules, MHC class II molecules, non-classical MHC molecules (such as HLA-G, HLA-E), MICA / B, ULBP1-6, IL6, IL6 receptor, IL1 receptor, RANKL, TGF-β1, PD1, PD-L1, CTLA4, Tim3, LAG3, Siglec-15, TIGIT, CD47, IL4RA, CD94 / NKG2A, CXCR1 / 2, CXCL8, CCR2 / CCR5, CCR4, CXCR4, c-Rel, CCL2, CCL5, CCL20, CCL22, CSF-1, CCL2, CCL5 indoleamine-2,3-dioxygenase (IDO), or arginase 1 (ARG1).

[0373] In some embodiments, the target protein includes a target protein associated with a nervous system disease. As used herein, the term "nervous system disease associated" refers to a protein involved in a nervous system disease, particularly a protein in a neurodegenerative disease. In some embodiments, the target protein associated with a nervous system disease includes, for example, Tau, amyloid-β (Aβ), α-synuclein, mutant huntingtin (mHTT), α-synuclein, TAR RNA binding protein (TARDBP) and FUS RNA binding protein (FUS).

[0374] In some embodiments, the target protein includes a target protein associated with viral infection. The target protein associated with viral infection includes, for example, HBV encoded X protein (HBx), HBV DNA polymerase, HBV capsid glycoprotein, HIV-1 reverse transcriptase, HIV gp120, HCV NS3-4A protease, HCV RNA polymerase, HCV envelope protein, EBV DNA polymerase, EBV EBNA1, coronavirus RNA synthetase, coronavirus spike protein, coronavirus envelope protein, coronavirus membrane protein, coronavirus nucleocapsid protein, RNA-dependent RNA polymerase (RdRp), such as SARS-CoV-2 RNA-dependent RNA polymerase, herpesvirus DNA and RNA polymerase, herpesvirus capsid glycoprotein, CMV DNA polymerase, CMV capsid glycoprotein, RSV surface protein, RSV capsid protein, RSV RNA polymerase, influenza virus RNA polymerase, influenza virus envelope protein, HPV DNA polymerase, HPV capsid protein. In another embodiment, the target protein is a viral protein, such as HBV surface antigen, HBeAg, HBV polymerase protein, HIV Gag protein, HIV Env protein, etc., whose function is believed to be important for viral replication, amplification and function and the progression of viral diseases.

[0375] In some embodiments, the target protein comprises a target protein associated with an autoantigen.A variety of autoantigens associated with autoimmune diseases are known in the art. For example, autoantigens associated with type 1 diabetes mellitus, such as islet cell antigen (ICA), insulin (IAA), glutamic acid decarboxylase 65 (GAD65), and insulinoma antigen-2 (IA-2); autoantigens associated with rheumatoid arthritis (RA), such as citrullinated protein / peptide antibodies, heterogeneous nuclear ribonucleoprotein A2 / B1, aldolase, alpha-enolase, calreticulin, heat-activated protein (HSP60), BiP, PGK1, stress-induced phosphoprotein 1, and FUSE-BP1 / 2; autoantigens associated with systemic lupus erythematosus (SLE), such as deoxyribonucleoprotein, SmD1 and SmD3, Clq, sore anticoagulant (LA), cardiolipin (CL), β2 glycoprotein I (β2GP I), prothrombin (PT), and phosphatidylserine (PS); and autoantigens associated with systemic sclerosis (SLE). Sclerosis (SSc) / scleroderma (SD)-related autoantigens such as Scl-70, SSA, Ro52; antigens related to autoimmune liver disease such as mitochondrial antigens, Sp100, PML, gp210, p62; autoantigens related to myasthenia gravis such as acetylcholine receptor; autoantigens related to central nervous system autoimmune diseases (limbic encephalitis, encephalomyelitis, cerebellar ataxia) such as voltage-gated potassium channel (VGKC) complex, voltage-gated calcium channel receptor, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor, γ-aminobutyric acid-B (GABAB) receptor, glycine receptor; autoantigens related to multiple sclerosis such as leukemia, myasthenia gravis, leukemia, and multiple sclerosis. Autoantigens related to polymyositis (PM) and dermatomyositis (DM) include Jo-1, Mi-2, PM-Scl, and Ro-52; autoantigens related to gluten-sensitive enteropathy include endomysial antibodies (EMA) and tissue transglutaminase (tTG); autoantigens related to anti-NMDAR antibody encephalitis include N-methyl-D-aspartate receptor; autoantigens related to neuromyelitis optica (NMO) include aquaporin 4 (AQP4); and autoantigens related to reproduction include ovarian antigens and sperm antigens.

[0376] In some embodiments, the pathogenic protein is a target protein associated with a metabolic disease. The target proteins related to metabolic diseases include, for example, target proteins for atherosclerosis (AS), including but not limited to CD36, low-density lipoprotein receptor (LDLR), ChemR23 (CMKLR1), and mitochondrial dehydrogenase ALDH4A1 (target protein); target proteins for type 2 diabetes, including but not limited to RalGAPα1 and dipeptidyl peptidase IV (DPP4); target proteins for non-alcoholic fatty liver disease, including but not limited to TMEM16A and VAMP3; target proteins for tumor glucose metabolism, including but not limited to hexokinase (HK), glucose transporter 1 (GLUT1), glucose transporter 4 (GLUT4), phosphoglycerate dehydrogenase, and lactate dehydrogenase; target proteins for tumor lipid metabolism, including but not limited to ATP citrate lyase (ACLY), fatty acid synthase (FAS), and ATP citrate lyase (ACLY). synthase, FASN), cholesterol esterase (acetyl-CoA acetyltransferase 1, ACAT1).

[0377] In some embodiments, the targeting domain specifically targets a target protein other than the following target proteins: TCR, HLA-I, MICA, and MICB, and the viral endoplasmic reticulum-resident protein is any viral endoplasmic reticulum-resident protein described in the present disclosure.

[0378] In some embodiments, the targeting domain specifically targets at least two of the following target proteins: TCR, HLA-I, MICA, MICB, and the viral endoplasmic reticulum-resident protein is any viral endoplasmic reticulum-resident protein described in the present disclosure.

[0379] In some embodiments, the targeting domain specifically targets TCR, HLA-I, MICA, or MICB, and the viral endoplasmic reticulum-resident protein is not adenoviral E3-K19.

[0380] 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 an amino acid sequence as shown in SEQ ID NO: 143 (IgG4 hinge) or SEQ ID NO: 141 ((Gly4Ser)2).

[0381] 3. Protein degradation pathway member binding domain

[0382] The chimeric protein constructs provided by the present disclosure may also comprise a protein degradation pathway member binding domain. As used herein, a "protein degradation pathway member binding domain" refers to any portion that is capable of directly or indirectly binding to a member of a protein degradation pathway.

[0383] The protein degradation pathway can be any pathway that can mediate protein degradation in cells. Known protein degradation pathways include, but are not limited to, ubiquitination-proteasome pathways, endosome-lysosome pathways, and autophagy degradation pathways.

[0384] Members of the ubiquitination-proteasome pathway include, for example, E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, E3 ubiquitin ligases, and the proteasome.

[0385] Examples of E1 ubiquitin-activating enzymes include, e.g., UBA1, UBA2, UBA3, UBA5, UBA6, UBA7, ATG7, NAE1, and SAE1.

[0386] Examples of E2 ubiquitin-conjugating enzymes include, e.g., hCdc34, Ubc-Uev1A, UBE2A, UBE2B, UBE2C, UBE2D1, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3, UBE2F, UBE2G1, UBE2G2, UBE2H, UBE2I, UBE2J1, UBE2J2, UBE2K, UBE2L3, UBE2L6, UBE2M, UBE2N, UBE2O, UBE2Q1, UBE2Q2, UBE2R1 (CDC34), UBE2R2, UBE2S, UBE2T, UBE2U, UBE2V1, UBE2V2, UBE2W, UBE2Z, ATG3, BIRC6, and UFC1.

[0387] Examples of E3 ubiquitin ligases include, for example, von Hippel–Lindau (VHL), Cereblon (CRBN), inhibitor of apoptosis protein (IAP), Kelch-like ECH-associated protein 1 (Keap1), RNF4, RNF114, MDM2, LUBAC, FBW7, Met30, HECT, SKP2, beta TRCP1, HUWEI, TRAF6, SMURF1, and E6AP. In certain embodiments, examples of E3 ubiquitin ligases include, for example, E3A, mdm2, Anaphase-promoting complex(APC),UBR5(EDD1),SOCS / BC-box / eloBC / CUL5 / RING,LNXp80,CBX4,CBLL1,HACE1,HECTD1,HECTD2,HECTD3,HECTD4,HECW1,HECW2,HERC1,HERC2,HERC3,HERC4,HERC5,HERC6,HUWE1,ITCH,NEDD4, NEDD4L, PPIL2, PRPF19, PIAS1, PIAS2, PIAS3, PIAS4, RANBP2, RNF4, RBX1, SMURF1, SMURF2, STUB1, TOPO RS, TRIP12, UBE3A, UBE3B, UBE3C, UBE3D, UBE4A, UBE4B, UBOX5, UBR5, VHL, WWP1, WWP2, Parkin, and MKRN1.

[0388] Members of the endosomal-lysosomal pathway include, for example, AP-1, AP-2, AP-3, endosomes, lysosomes, HOPS, ESCRT, GASP, BLOC-1, ESCRT, Retromer, ESCRT, sortingnexin, Dapper2, SNX4, Pincher, Rap1-PDZ-GEF1, clathrin, C3G / CrkL / Shp2 / Gab2, etc.

[0389] 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.

[0390] In some embodiments, the protein degradation pathway member binding domain is a VHL binding domain. In some embodiments, the VHL binding domain comprises the amino acid sequence of SEQ ID NO: 163 (DRHDS(p)GLDS(p)M) or SEQ ID NO: 164 (ALAPYIP).

[0391] In some embodiments, the protein degradation pathway member binding domain is a Keap1 binding domain. In some embodiments, the Keap1 binding domain comprises the amino acid sequence of SEQ ID NO: 165 (LDPETGEYL).

[0392] In some embodiments, the protein degradation pathway member binding domain is an E3 ubiquitin ligase binding domain comprising the amino acid sequence of SEQ ID NO: 166 (DRHDSGLDSM).

[0393] In some embodiments, the protein degradation pathway member binding domain is a CMA binding domain. In some embodiments, the CMA binding domain comprises the amino acid sequence of SEQ ID NO: 167 (KFERQ). In some embodiments, the CMA binding domain comprises the amino acid sequence of SEQ ID NO: 168 (KFERQKILDQRFFE).

[0394] In some embodiments, the protein degradation pathway member binding domain is a proteasome binding domain. In some embodiments, the proteasome binding domain is selected from the group consisting of: yeast Rad23 (e.g., S. cerevisiae Rad23), the ubiquitin-like (UbL) domain of human Rad23b (hHR23b), HPV E7, and the proteasome binding domain of ankyrin. In some embodiments, the proteasome binding domain comprises amino acids 1-77 of yeast Rad23. In some embodiments, the proteasome binding domain comprises amino acids 1-83 of human Rad23b.

[0395] The inventors of the present application have found that compared with the basic TPD design (Targeted protein binding domain-Transmembrane domain-ER retention domain, TBD-TMD-ERD), chimeric protein constructs containing protein degradation pathway member binding domains (ligand for E3 Ligase (E3L), ligand for E2 Ubiquitin-conjugating enzyme (E2L) or ligand for lysosome (LL)) (for example, those described above) have significantly improved degradation effects on target proteins. The functional structure of viral ER-resident proteins has different binding abilities to the ERAD reverse transporter complex, and the natural structure also interacts differently with the ER-resident E3 ligase. At the same time, it also competes with the normal ERAD degradation pathway of misfolded proteins in the endoplasmic reticulum. Therefore, the natural TMD-ERD domain has different abilities to ubiquitinate and degrade target proteins. In order to further improve the degradation efficiency of target proteins, the introduction of ubiquitin-proteasome system (UPS) ligand domain and / or autophagy-lysosome pathway (ALP) ligand domain can further ubiquitinate the target proteins retained in the endoplasmic reticulum and then achieve proteasomal degradation or target protein degradation through lysosomes.

[0396] In some embodiments, the targeting domain or ERAD machinery protein binding domain of the chimeric protein construct is connected to the protein degradation pathway member binding domain via a hinge or linker. In some embodiments, the hinge comprises the amino acid sequence shown in SEQ ID NO: 147 (IgG4 hinge) or SEQ ID NO: 144 ((Gly4Ser)2).

[0397] 4. TPD chimeric protein construct

[0398] In some embodiments, the chimeric protein constructs provided herein comprise a targeting protein binding domain that targets a TCR, an adenovirus E3-19K, and one or more protein degradation pathway member binding domains described above. In some embodiments, the chimeric protein constructs provided herein comprise a targeting protein binding domain that targets a TCR, an adenovirus E3-19K, and an E3 ubiquitin ligase binding domain. In some embodiments, the E3 ubiquitin ligase binding domain comprises the amino acid sequence shown in SEQ ID NO: 162 (DRHDSGLDSMGSGSGALAPYIP).

[0399] In some embodiments, the chimeric protein construct provided by the present disclosure comprises a targeting protein binding domain that targets a TCR and a transmembrane domain or a functional variant thereof and an endoplasmic reticulum retention domain or a functional variant thereof of a viral endoplasmic reticulum resident protein other than adenovirus E3-19K. The viral endoplasmic reticulum resident protein can be at least one selected from the group consisting of HCMV glycoprotein US2, US11, US3, US10, US6, HSV ICP47, CPXV12, BHV UL49.5, EBV BNFL2a, HCMV UL16, UL141, UL142, HIV Nef, HIV Vpu, HHV-7U21, HHV-8KK3, HHV-8KK5, MHV-68MK3, HTLV-1p12, and vaccinia virus protein CPXV203.

[0400] In some embodiments, the chimeric protein construct provided by the present disclosure comprises a targeting protein binding domain that targets the above-mentioned target protein (e.g., TCR) and a transmembrane domain or a functional variant thereof and an endoplasmic reticulum retention domain or a functional variant thereof of HCMV US2 and / or US11.

[0401] In some embodiments, the chimeric protein construct provided by the present disclosure comprises a targeting protein binding domain that targets the above-mentioned target protein (e.g., TCR) and the transmembrane domain or a functional variant thereof and the endoplasmic reticulum retention domain or a functional variant thereof of HCMV US3.

[0402] In some embodiments, the chimeric protein construct provided by the present disclosure comprises a targeting protein binding domain that targets the above-mentioned target protein (e.g., TCR) and the transmembrane domain or a functional variant thereof and the endoplasmic reticulum retention domain or a functional variant thereof of HCMV US10.

[0403] In some embodiments, the chimeric protein constructs provided by the present disclosure comprise a targeting protein binding domain that targets the above-mentioned target protein (e.g., TCR) and a transmembrane domain or a functional variant thereof of HCMV US6, HSV ICP47, CPXV012, EBV BNFL2a and / or BHV UL49.5 and an endoplasmic reticulum retention domain or a functional variant thereof.

[0404] In some embodiments, the chimeric protein construct provided by the present disclosure comprises a targeting protein binding domain that targets the above-mentioned target protein (e.g., TCR) and the transmembrane domain or a functional variant thereof and the endoplasmic reticulum retention domain or a functional variant thereof of HHV-7US21.

[0405] In some embodiments, the chimeric protein constructs provided by the present disclosure comprise a targeting protein binding domain that targets the above-mentioned target protein (e.g., TCR) and a transmembrane domain or a functional variant thereof and an endoplasmic reticulum retention domain or a functional variant thereof of HCMV UL16, UL141 and / or UL142.

[0406] In some embodiments, the chimeric protein constructs provided by the present disclosure comprise a targeting protein binding domain that targets the above-mentioned target protein (e.g., TCR) and a transmembrane domain or a functional variant thereof and an endoplasmic reticulum retention domain or a functional variant thereof of HIV Nef, HIV Vpu, HHV-7U21, HHV-8KK3, HHV-8KK5, MHV-68MK3 and / or HTLV-1p12.

[0407] In some embodiments, the chimeric protein construct provided by the present disclosure comprises a targeting protein binding domain that targets the above-mentioned target protein (e.g., TCR) and a transmembrane domain or a functional variant thereof and an endoplasmic reticulum retention domain or a functional variant thereof of the Cowpox Virus protein CPXV203.

[0408] In some embodiments, the present disclosure provides a chimeric protein construct comprising a targeting protein binding domain that is not a targeting TCR and adenovirus E3-19K.

[0409] In some embodiments, the chimeric protein construct further comprises one or more protein degradation pathway member binding domains as described above.

[0410] 5. Co-expression

[0411] In some embodiments, the chimeric protein constructs provided herein and at least one co-expression moiety are co-expressed. Any suitable method for co-expression may be used. For example, the chimeric protein construct (or a portion thereof) and the at least one co-expression moiety may be connected by a cleavable linker, such that upon cleavage of the linker, the chimeric protein construct and the co-expression moiety can be co-expressed.

[0412] In some embodiments, the chimeric protein constructs provided herein and at least one co-expression moiety are linked, for example, via a self-cleavable linker. In some embodiments, the self-cleavable linker is a cleavable peptide; for example, a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, a F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide.

[0413] In some preferred embodiments, the T2A comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.144, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of T2A is as shown in SEQ ID NO.144.

[0414] In some preferred embodiments, the amino acid sequence of the GSG-T2A peptide is shown as SEQ ID NO.145.

[0415] In some preferred embodiments, the P2A comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.146, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the P2A is as shown in SEQ ID NO.146.

[0416] In some preferred embodiments, the amino acid sequence of the GSG-P2A peptide is shown as SEQ ID NO.147.

[0417] In some preferred embodiments, the E2A comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 148, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the E2A is as shown in SEQ ID NO. 148.

[0418] In some preferred embodiments, the amino acid sequence of the GSG-E2A peptide is shown as SEQ ID NO.149.

[0419] In some preferred embodiments, the F2A comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 150, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or 99% or more identity; the amino acid sequence of F2A is shown in SEQ ID NO. 150.

[0420] In some preferred embodiments, the amino acid sequence of the GSG-F2A peptide is shown as SEQ ID NO.151.

[0421] The coexpression portion provided herein can be any protein or polypeptide with a biological function. Depending on the biological function desired to be achieved, a suitable coexpression portion can be selected. For example, in order to reduce the immunogenicity or antigen presentation of the cell, a protein that can degrade or reduce MHC class I or class II molecules can be selected as a coexpression portion. For another example, in order to enable the cell to recognize the target protein, a binding domain (such as a chemokine receptor or a chimeric antigen receptor CAR) that recognizes or binds to the target protein can be selected as a coexpression portion. For another example, in order to increase immunostimulatory activity, an immunostimulatory molecule can be selected as a coexpression portion.

[0422] In some preferred embodiments, examples of the co-expressed moieties include, but are not limited to, complete viral ER resident glycoproteins (e.g., HCMV US2, US3, US11, US10, adenovirus E3-K1 9, HCMV US6, HSV ICP47), chimeric antigen receptors (CARs), functional T cell receptors (TCRs), chemokine receptors (e.g., CCR4, CCR5, CCR6, CCR7, CCR9, CCR2b, CXCR1, CXCR2, and CXCR4), NK cell activating receptors (e.g., NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, K IR3DS1, natural cytotoxicity receptor, TRAIL, DNAM-1, CD16a, 2B4, NTB-A, CRACC and NKp80), CNK signal transduction components, cytokines, CD7, immunostimulatory molecules (e.g., one or more of TNF-a, IFN-β, IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18 and granulocyte macrophage colony stimulating factor), etc.

[0423] i) Intact viral ER-resident glycoprotein

[0424] In some embodiments, the co-expressed moiety is an intact viral ER-resident glycoprotein, including but not limited to HCMV US2, US3, US11, US10, adenovirus E3-K19, HCMV US6, and HSV ICP47.

[0425] In some preferred embodiments, the full-length sequence of the HCMV glycoprotein US2 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.82, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US2 is as shown in SEQ ID NO.82.

[0426] In some preferred embodiments, the full-length sequence of US2 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.110, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of US2 is as shown in SEQ ID NO.110.

[0427] In some preferred embodiments, the full-length sequence of the HCMV glycoprotein US3 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.85, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US3 is as shown in SEQ ID NO.85.

[0428] In some preferred embodiments, the full-length sequence of US3 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.113, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of US3 is as shown in SEQ ID NO.113.

[0429] In some preferred embodiments, the full-length sequence of the HCMV glycoprotein US11 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.88, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US11 is as shown in SEQ ID NO.88.

[0430] In some preferred embodiments, the full-length sequence of US11 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.116, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of US11 is as shown in SEQ ID NO.116.

[0431] In some preferred embodiments, the full-length sequence of the HCMV glycoprotein US10 comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.91, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of the HCMV glycoprotein US10 is as shown in SEQ ID NO.91.

[0432] In some preferred embodiments, the full-length sequence of the adenovirus E3-19K comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.94, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of the adenovirus E3-19K is as shown in SEQ ID NO.94.

[0433] 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 an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of HCMV US6 is as shown in SEQ ID NO.100.

[0434] In some preferred embodiments, the full-length sequence of HSV ICP47 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.102, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical, and preferably has the activity of degrading MHC class I or class II molecules; the amino acid sequence of the full-length sequence of HSV ICP47 is as shown in SEQ ID NO.102.

[0435] ii) Chimeric Antigen Receptor (CAR)

[0436] In certain embodiments, the co-expression portion is a chimeric antigen receptor (CAR). The CAR comprises a target protein binding domain (e.g., an extracellular recognition domain targeting a tumor), a transmembrane domain, and an immunoreceptor activation signaling domain (ITAM) (also referred to as an "intracellular signaling domain"). In certain embodiments, the CAR may further comprise a costimulatory domain. In certain embodiments, a hinge or joint is included between the extracellular recognition domain, the transmembrane domain, and / or the intracellular signaling domain targeting a tumor.

[0437] In some embodiments, the tumor-targeting extracellular recognition domain is selected from a tumor antigen-specific binding domain, a tumor microenvironment target antigen binding domain, and / or a tumor microenvironment-targeting chemotactic receptor.

[0438] In some preferred embodiments, the tumor-targeting extracellular recognition domain is selected from an antibody or a functional fragment thereof, a TCR or a combination thereof that can target and recognize tumor-associated antigens; the functional fragment of the antibody is selected from Fd, Fv, Fab, Fab', F(ab')2, Fv (scFv), a single-chain antibody (scFv) or a nanobody, a double-chain antibody, a three-chain antibody and a four-chain antibody.

[0439] In some preferred embodiments, the transmembrane domain of the CAR described herein can be derived from any membrane-bound protein or transmembrane protein, including but not limited to BAFFR, BLAME (SLAMF8), CD2, CD3ε, CD4, CD5, CD8, CD9, CD11a (CD18, ITGAL, LFA-1), CD11b, CD11c, CD11d, CD16, CD19, CD22, CD27, CD28, CD29, CD33, CD37, CD40, CD45, CD49a, CD49d, C D49f, CD64, CD80, CD84, CD86, CD96(Tactile), CD100(SEMA4D), CD103, CD134, CD137(4-1BB), CD150(IPO-3, SLAMF1, SLA M), CD154, CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(Ly9), CD244(2B4, SLAMF4), CD278(ICOS), CEACAM1, CRT AM, GITR, HYEM (LIGHTR), IA4, IL2Rβ, IL2Rγ, IL7R α, ITGA1, ITGA4, ITGA6, ITGAD, ITGAE, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIR, LTBR, ​​OX40, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG / Cbp, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, α, β, or ζ chain of the T cell receptor, TNFR2, VLA1, and VLA-6.

[0440] In some preferred embodiments, the transmembrane domain of the CAR is selected from the group consisting of an NK cell activating receptor transmembrane domain, a DAP10 transmembrane domain, a DAP12 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, and a BTLA transmembrane domain, and combinations thereof.

[0441] In some preferred embodiments, the transmembrane domain of the CAR is selected from the group consisting of a CD8 transmembrane domain, an α and / or β chain transmembrane domain of a T cell receptor, a CD28 transmembrane domain, a CD3ε transmembrane domain, a CD45 transmembrane domain, a CD4 transmembrane domain, a CD5 transmembrane domain, a CD8 transmembrane domain, a CD9 transmembrane domain, a CD16 transmembrane domain, a CD22 transmembrane domain, a CD33 transmembrane domain, a CD37 transmembrane domain, a CD64 transmembrane domain, a CD80 transmembrane domain, a CD86 transmembrane domain, a CD134 transmembrane domain, a CD137 transmembrane domain, a CD154 transmembrane domain, a GITR transmembrane domain, and combinations thereof.

[0442] In some preferred embodiments, the immunoreceptor activation signaling domain (ITAM) is derived from the intracellular activation signaling domain of an immunoreceptor; preferably, the immunoreceptor is selected from TCRζ, CD2, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, FcRγ, CD66d, FcαRI, FcγRI, FcγRII, FcγRIII, Dectin-1, CLEC-1, CD72, CD79A, CD79B; preferably, the immunoreceptor activation signaling domain (ITAM) is fused with an NK cell signal transducer or a functional variant thereof; preferably, the immunoreceptor is CD3ζ.

[0443] In some preferred embodiments, the immunoreceptor activation signaling domain (ITAM) is derived from intracellular signaling domains such as CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI CD66d, DAP10 and DAP12.

[0444] In some preferred embodiments, the intracellular signaling domain of the CAR includes the intracellular signaling domain and / or the co-stimulatory signaling domain of an NK cell activating receptor.

[0445] 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, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD16, CD27, CD28, CD30, CD40, CD38, CD35, CD79A, CD79B, CDS, ICAM-1, LFA-1, (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GI TR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL 7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD1 1b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, NCR, DAP10, DAP12, TNFR2, TRANCE / RANKL, DNAM1(CD22 6), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100SEMA4D), CD69, SLAMF 6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, ligand that specifically binds to CD83, CARD11, FcRa, FcRp, FcRy, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, Wnt, OX40, ROR2, Ryk, SLAMF1, Slp76, pTa, TCRa, TCRp, TRIM, ZAP70, PTCH2.More preferably, the costimulatory signaling domain can be selected from the group consisting of NKG2D intracellular signaling domain, DAP10 intracellular signaling domain, DAP12 intracellular signaling domain, NCR intracellular signaling domain, CD28 intracellular signaling domain, 4-1BB intracellular signaling domain, OX40 intracellular signaling domain, and ICOS intracellular signaling domain.

[0446] In some embodiments, the hinge and / or transmembrane domain of CAR described herein provide the cell surface presentation of the extracellular domain of CAR. The hinge of CAR described herein can be derived from any membrane-bound protein or transmembrane protein, including but not limited to BAFFR, BLAME (SLAMF8), CD2, CD3ε, CD4, CD5, CD8, CD9, CD11a (CD18, ITGAL, LFA-1), CD11b, CD11c, CD11d, CD16, CD19, CD22, CD27, CD28, CD29, CD33, CD37, CD40, CD45, CD49a, CD49d, CD49f, CD6 4. CD80, CD84, CD86, CD96 (Tactile), CD100 (SEMA4D), CD103, CD134, CD137 (4-1BB), CD150 (IPO-3, SLAMF1, SLAM), C D154, CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(Ly9), CD244(2B4, SLAMF4), CD278(ICOS), CEACAM1, CRT AM, GITR, HYEM (LIGHTR), IA4, IL2R β, IL2R γ, IL7Ra, ITGA1, ITGA4, ITGA6, ITGAD, ITGAE, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIR, LTBR, ​​OX40, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG / Cbp, PSGL1, SLAMF6 (NTB-A, Ly108), SLAMF7, α, β or ζ chain of T cell receptor, TNFR2, VLA1 and VLA-6. In some embodiments, the hinge of CAR described herein includes the hinge region of CD8 α, the hinge region of human immunoglobulin (Ig) or a glycine-serine-rich sequence.

[0447] In some preferred embodiments, the linker is a flexible linker; preferably, the flexible linker comprises the amino acid sequence shown (Gly(x)Ser(y))n, wherein n is an integer from 1 to 10, and x and y are independently integers from 0 to 10, with the proviso that x and y are not both 0; more preferably, the linker comprises the amino acid sequence shown in SEQ ID NO.140 or the amino acid sequence shown in SEQ ID NO.141.

[0448] In some preferred embodiments, the hinge is an IgG1 hinge or an IgG4 hinge.

[0449] In some preferred embodiments, the IgG1 hinge comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.142, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the IgG1 hinge is as shown in SEQ ID NO.142.

[0450] In some preferred embodiments, the IgG4 hinge comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.143, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the IgG4 hinge is as shown in SEQ ID NO.143.

[0451] iii) CNK receptors

[0452] In certain embodiments, the co-expressed portion is a CNK receptor. The CNK receptor comprises a chimeric NK activating receptor component and a chimeric NK signal transduction component (also referred to as a "CNK signal transduction component"). The chimeric NK activating receptor component may comprise at least an NK cell activating receptor or a functional variant thereof.

[0453] NK cell activating receptor

[0454] In some embodiments, the NK cell activating receptor comprises: (a) an NK cell activating receptor extracellular domain (ED) or a functional variant thereof, (b) an NK cell activating receptor transmembrane domain (TMD) or a functional variant thereof, and (c) an NK cell activating receptor intracellular domain (ICD) or a functional variant thereof; Optionally, a hinge or joint is included between the NK cell activating receptor extracellular domain or a functional variant thereof, the NK cell activating receptor transmembrane domain or a functional variant thereof and / or the NK cell activating receptor intracellular domain or a functional variant thereof. A detailed description of NK cell activating receptors and CNK signaling switching components can be found in, for example, US 2020 / 0308248A1, the entire contents of which are incorporated herein by reference.

[0455] The NK cell activating receptor is selected from NKG2D, NKG2C, NKG2E, NKG2F, NKG2H, CD94, KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, natural cytotoxicity receptors (NCR), TRAIL, DNAM-1, signaling lymphocytic activation molecule (SLAM) family molecule 2B4 (also known as CD244), DNAX attachment molecule 1 (DNAM-1, also known as CD226), CD16a, 2B4, NTB-A, CRACC (CS1) and NKp80; wherein the natural cytotoxicity receptor includes NKp46 (also known as NCR1 or CD335), NKp44 (also known as NCR2 or CD336) and NKp30 (also known as NCR3 or CD337). In some embodiments, the natural cytotoxicity receptor is selected from NKp46, NKp44, and NKp30.

[0456] In some preferred embodiments, the NK cell activating receptor is a NK cell activating receptor of mammalian origin; preferably, the mammal is selected from humans, primates, mice, horses, cows, sheep, goats, cats, pigs, dogs, llamas, alpacas, elephants, squirrels, and guinea pigs.

[0457] 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.

[0458] In some preferred embodiments, the NK cell activating receptor is a human NK cell activating receptor; preferably, the NK cell activating receptor is a recombinant NK cell activating receptor comprising different human NK cell activating receptor domains.

[0459] In some preferred embodiments, the NK cell activating receptor is a murine NK cell activating receptor; preferably, the NK cell activating receptor is a recombinant NK cell activating receptor comprising different murine NK cell activating receptor domains.

[0460] 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.

[0461] In some preferred embodiments, the extracellular domain of the NK cell activating receptor is the extracellular domain of a human or mouse NK cell activating receptor.

[0462] In some preferred embodiments, the transmembrane domain of the NK cell activating receptor is a transmembrane domain of a human or mouse NK cell activating receptor.

[0463] In some preferred embodiments, the intracellular domain of the NK cell activating receptor is the intracellular domain of a human or mouse NK cell activating receptor.

[0464] In some preferred embodiments, the functional variant of the NK cell activating receptor is selected from a mutant of the NK cell activating receptor, a wild-type fusion protein, or a fusion protein of the wild-type and mutant types.

[0465] In some preferred embodiments, the extracellular domain of human NKG2D comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.37, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the extracellular domain of human NKG2D is as shown in SEQ ID NO.37.

[0466] In some preferred embodiments, the full-length sequence of human NKG2D comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.38, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKG2D is as shown in SEQ ID NO.38.

[0467] In some preferred embodiments, the extracellular domain of mouse NKG2D comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.39, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the extracellular domain of mouse NKG2D is as shown in SEQ ID NO.39.

[0468] In some preferred embodiments, the full-length sequence of mouse NKG2D comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO:40, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of mouse NKG2D is as shown in SEQ ID NO.40.

[0469] In some preferred embodiments, the full-length sequence of human-mouse recombinant NKG2D comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO:41, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human-mouse recombinant NKG2D is as shown in SEQ ID NO.41.

[0470] In some preferred embodiments, the full-length sequence of human NKG2C comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.42, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKG2C is as shown in SEQ ID NO.42.

[0471] In some preferred embodiments, the full-length sequence of human NKG2E comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 43, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKG2E is as shown in SEQ ID NO.43.

[0472] In some preferred embodiments, the full-length sequence of human NKG2F comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 44, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKG2F is as shown in SEQ ID NO.44.

[0473] In some preferred embodiments, the full-length sequence of human CD94 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 45, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence is as shown in SEQ ID NO. 45.

[0474] In some preferred embodiments, the full-length sequence of human KIR2DL4 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 46, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human KIR2DL4 is as shown in SEQ ID NO.46.

[0475] In some preferred embodiments, the full-length sequence of human KIR2DS1 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 47, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human KIR2DS1 is as shown in SEQ ID NO.47.

[0476] In some preferred embodiments, the full-length sequence of human KIR2DS2 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 48, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human KIR2DS2 is as shown in SEQ ID NO.48.

[0477] In some preferred embodiments, the full-length sequence of human KIR2DS4 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 49, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human KIR2DS4 is as shown in SEQ ID NO.49.

[0478] In some preferred embodiments, the full-length sequence of human KIR3DS1 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 50, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human KIR3DS1 is as shown in SEQ ID NO.50.

[0479] In some preferred embodiments, the full-length sequence of human NKp46 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.51, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKp46 is as shown in SEQ ID NO.51.

[0480] In some preferred embodiments, the full-length sequence of human NKp44 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.52, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKp44 is as shown in SEQ ID NO.52.

[0481] In some preferred embodiments, the full-length sequence of human NKp30 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.53, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKp30 is as shown in SEQ ID NO.53.

[0482] In some preferred embodiments, the full-length sequence of human DNAM1 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 54, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human DNAM1 is as shown in SEQ ID NO.54.

[0483] In some preferred embodiments, the full-length sequence of human TRAIL comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 55, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human TRAIL is as shown in SEQ ID NO.55.

[0484] In some preferred embodiments, the full-length sequence of human CD16a comprises an amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO: 56, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length human CD16a is as shown in SEQ ID NO. 56.

[0485] In some preferred embodiments, the full-length sequence of human 2B4 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 57, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human 2B4 is shown in SEQ ID NO. 57.

[0486] In some preferred embodiments, the full-length sequence of human NTB-A comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 58, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NTB-A is as shown in SEQ ID NO. 58.

[0487] In some preferred embodiments, the full-length sequence of human CRACC comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 59, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human CRACC is shown in SEQ ID NO. 59.

[0488] In some preferred embodiments, the full-length sequence of human NKp80 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 60, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human NKp80 is as shown in SEQ ID NO.60.

[0489] CNK signal adapter components

[0490] In some embodiments, the CNK signal adapter component comprises at least (i) an NK cell signal converter (adaptor) (e.g., DAP10 or DAP12) or a functional variant thereof. In some embodiments, the NK cell signal converter comprises: (a) an NK cell signal converter extracellular domain (ED) or a functional variant thereof, (b) an NK cell signal converter transmembrane domain (TMD) or a functional variant thereof, and (c) an NK cell signal converter intracellular domain (ICD) or a functional variant thereof; optionally, a hinge or linker is included between the NK cell signal converter extracellular domain or a functional variant thereof, the NK cell signal converter transmembrane domain or a functional variant thereof, and / or the NK cell signal converter intracellular domain or a functional variant thereof.

[0491] In some preferred embodiments, the NK cell signal transducer is a NK cell signal transducer of mammalian origin; preferably, the mammal is selected from humans, primates, mice, horses, cows, sheep, goats, cats, pigs, dogs, llamas, alpacas, elephants, squirrels, and guinea pigs.

[0492] 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.

[0493] In some preferred embodiments, the NK cell signal transducer is a human NK cell signal transducer; preferably, the NK cell signal transducer is a recombinant NK cell signal transducer comprising different human NK cell signal transducer domains.

[0494] In some preferred embodiments, the NK cell signal transducer is a murine NK cell signal transducer; preferably, the NK cell signal transducer is a recombinant NK cell signal transducer comprising different murine NK cell signal transducer domains.

[0495] 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.

[0496] In some preferred embodiments, the extracellular domain of the NK cell signal transducer is the extracellular domain of a human or mouse NK cell signal transducer.

[0497] In some preferred embodiments, the transmembrane domain of the NK cell signal transducer is a transmembrane domain of a human or mouse NK cell signal transducer.

[0498] In some preferred embodiments, the intracellular domain of the NK cell signal transducer is the intracellular domain of a human or mouse NK cell signal transducer;

[0499] The NK cell signal converter in the CNK signal adapter component is DAP10 or DAP12.

[0500] 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.

[0501] In some preferred embodiments, the CNK signal transduction component further comprises (ii) an immunoreceptor activation signaling domain (ITAM) and / or (iii) a T cell co-stimulatory signaling domain.

[0502] In some preferred embodiments, a hinge or linker is contained between the NK cell signal transducer or a functional variant thereof, the immunoreceptor activation signaling domain (ITAM) and / or the T cell co-stimulatory signaling domain; preferably, the NK cell signal transducer or a functional variant thereof is fused with the immunoreceptor activation signaling domain (ITAM) domain.

[0503] In some preferred embodiments, the immunoreceptor activation signaling domain (ITAM) is derived from the intracellular activation signaling domain of an immunoreceptor; preferably, the immunoreceptor is selected from TCRζ, CD2, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, FcRγ, CD66d, FcαRI, FcγRI, FcγRII, FcγRIII, Dectin-1, CLEC-1, CD72, CD79A, CD79B; preferably, the immunoreceptor activation signaling domain (ITAM) is fused with an NK cell signal transducer or a functional variant thereof; preferably, the immunoreceptor is CD3ζ.

[0504] In some preferred embodiments, the T cell costimulatory signaling domain is derived from the intracellular signaling domain of a costimulatory molecule.

[0505] In some preferred embodiments, the full-length sequence of human DAP10 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.61, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human DAP10 is as shown in SEQ ID NO.61.

[0506] In some preferred embodiments, the full-length sequence of human DAP10 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.62, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human DAP10 is as shown in SEQ ID NO.62.

[0507] In some preferred embodiments, the transmembrane domain of human DAP10 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.63, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the transmembrane domain of human DAP10 is as shown in SEQ ID NO.63.

[0508] In some preferred embodiments, the full-length sequence of human DAP12 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.64, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the full-length sequence of human DAP12 is as shown in SEQ ID NO.64.

[0509] In some preferred embodiments, the transmembrane domain of human DAP12 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.65, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the transmembrane domain of human DAP12 is as shown in SEQ ID NO.65.

[0510] In some preferred embodiments, the transmembrane domain fusion protein of human DAP10 and human DAP12 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.66, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequences of the transmembrane domains of human DAP10 and human DAP12 are as shown in SEQ ID NO.66.

[0511] In some preferred embodiments, the human DAP10-DAP12 fusion protein sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.67, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP10-DAP12 fusion protein sequence is as shown in SEQ ID NO.67.

[0512] In some preferred embodiments, the human CD3zeta intracellular signaling domain sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.68, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human CD3zeta intracellular signaling domain sequence is as shown in SEQ ID NO.68.

[0513] In some preferred embodiments, the human DAP10-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.69, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP10-CD3zeta sequence is as shown in SEQ ID NO.69.

[0514] In some preferred embodiments, the human DAP12-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.70, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP12-CD3zeta sequence is as shown in SEQ ID NO.70.

[0515] In some preferred embodiments, the human DAP10-DAP12-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.71, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP10-DAP12-CD3zeta sequence is as shown in SEQ ID NO.71.

[0516] In some preferred embodiments, the human 41BB intracellular signaling domain sequence comprises an amino acid sequence having 80% or greater identity to the amino acid sequence shown in SEQ ID NO. 72, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity; the amino acid sequence of the human 41BB intracellular signaling domain sequence is shown in SEQ ID NO. 72.

[0517] 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 an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or 99% or more identity; the amino acid sequence of the human DAP10-41BB sequence is as shown in SEQ ID NO.73.

[0518] In some preferred embodiments, the human DAP10-41BB-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.74, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP10-41BB-CD3zeta sequence is as shown in SEQ ID NO.74.

[0519] 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 an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or 99% or more identity; the amino acid sequence of the human CD28 intracellular signaling domain sequence is as shown in SEQ ID NO. 75.

[0520] In some preferred embodiments, the human DAP10-CD28 sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.76, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP10-CD28 sequence is as shown in SEQ ID NO.76.

[0521] In some preferred embodiments, the human DAP10-CD28-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.77, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP10-CD28-CD3zeta sequence is as shown in SEQ ID NO.77.

[0522] 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 an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or 99% or more identity; the amino acid sequence of the human DAP12-41BB sequence is as shown in SEQ ID NO.78.

[0523] In some preferred embodiments, the human DAP12-41BB-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.79, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP12-41BB-CD3zeta sequence is as shown in SEQ ID NO.79.

[0524] In some preferred embodiments, the human DAP12-CD28 sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.80, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP12-CD28 sequence is as shown in SEQ ID NO.80.

[0525] In some preferred embodiments, the human DAP12-CD28-CD3zeta sequence comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO.81, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; the amino acid sequence of the human DAP12-CD28-CD3zeta sequence is as shown in SEQ ID NO.81.

[0526] In some preferred embodiments, the CNK signal transduction component comprises an amino acid sequence selected from SEQ ID NOs. 61 to 81.

[0527] In some preferred embodiments, the chimeric protein construct forms a multifunctional complex with the NK cell activating receptor and the CNK signaling transduction component, wherein the multifunctional complex comprises the amino acid sequence shown in SEQ ID NO. 152. In some preferred embodiments, the multifunctional complex comprises an amino acid sequence having 80% or greater identity to the amino acid sequence shown in SEQ ID NO. 152, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity, and more preferably an amino acid sequence having 98% or greater identity; the amino acid sequence of the multifunctional complex comprises a TCR antibody single-chain antibody shown in SEQ ID NO. 152.

[0528] 6. Nucleic acid molecules

[0529] In another aspect, the present disclosure also provides a chimeric nucleic acid construct encoding any chimeric protein construct described in the present application.

[0530] The nucleic acid molecule may include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with β-D-ribose configuration, α-LNA with α-L-ribose configuration (diastereomers of LNA), 2'-amino-LNA with 2'-amino functionalization and 2'-amino-α-LNA with 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and / or chimeras and / or combinations thereof. Exemplary DNA includes but is not limited to plasmid DNA (pDNA) and the like. Exemplary RNA includes but is not limited to mRNA, circular RNA, ccRNA.

[0531] In some preferred embodiments, the nucleic acid molecule is mRNA. mRNA is any RNA encoding at least one protein, and the encoded protein can be produced in vitro, in vivo, in situ or in vitro. It will be understood by those skilled in the art that, unless otherwise stated, the nucleic acid sequence set forth in this application can be listed as "T" in a representative DNA sequence, but in the case where the sequence represents RNA (e.g., mRNA), "T" will be replaced with "U". Therefore, any DNA disclosed and identified herein by a specific sequence identification number also discloses a corresponding RNA (e.g., mRNA) sequence complementary to the DNA, wherein each "T" of the DNA sequence is replaced by a "U".

[0532] In some preferred embodiments, the nucleic acid molecule comprises a nucleotide sequence that is 80% or more identical to the nucleotide sequence shown in SEQ ID NO. 154, preferably a nucleotide sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably a nucleotide sequence that is 98% or 99% or more identical.

[0533] In some preferred embodiments, the nucleic acid molecule is an in vitro synthesized mRNA. In some preferred embodiments, the in vitro synthesized mRNA has modifications, optionally including one or more modifications selected from the group consisting of: a 5'UTR, a 5'UTR, a poly A tail, a 5'-cap, and one or more modified nucleotides in the coding region.

[0534] i) Modification of nucleic acid molecules (e.g. mRNA)

[0535] Naturally occurring eukaryotic mRNA molecules may contain stabilizing elements, including but not limited to an untranslated region (UTR) at its 5'-end (5'UTR) and / or a UTR at its 3'-end (3'UTR), as well as other structural features, such as a 5'-cap structure, a 3'-poly A tail. Both the 5'UTR and the 3'UTR are typically transcribed from genomic DNA and are part of premature mRNA. The characteristic structures of mature mRNA (such as the 5'-cap structure and the 3'-poly A tail) are typically added to the transcribed (premature) mRNA during mRNA processing.

[0536] "5'UTR" refers to the region of an mRNA immediately upstream (i.e., 5') of the start codon (i.e., the first codon of the mRNA transcript translated by the ribosome) that does not encode a polypeptide. When an RNA transcript is generated, the 5'UTR may contain a promoter sequence. Such promoter sequences are known in the art.

[0537] "3'UTR" refers to the region of an mRNA immediately downstream (ie, 3') of the stop codon (ie, the codon of an mRNA transcript that indicates the termination of translation) that does not encode a polypeptide.

[0538] A "poly A tail" is a downstream region of an mRNA, for example, immediately downstream (i.e., 3') of the 3' UTR, comprising a plurality of consecutive adenosine monophosphates. A poly A tail may contain 10 to 300 adenosine monophosphates. For example, a poly A tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, the poly A tail contains 50 to 250 adenosine monophosphates. In a relevant biological context (e.g., in a cell or in vivo), the role of the poly A tail is to protect mRNA from enzymatic degradation (e.g., enzymatic degradation in the cytoplasm) and to facilitate transcription termination and / or export and translation of mRNA from the nucleus. The 3'-poly A tail is typically a section of adenine nucleotides added to the 3'-end of a transcribed mRNA. In some cases, it can contain up to about 400 adenine nucleotides. The length of the 3'-poly A tail can affect the stability of the mRNA molecule itself.

[0539] 5'-Capping of polynucleotides can be accomplished simultaneously during the in vitro transcription reaction using the following chemical RNA cap analogs to generate a 5'-guanosine cap structure: 3'-O-Me-m7G(5')ppp(5')G [ARCA cap], G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G (New England Biolabs, Ipswich, MA). 5'-Capping of modified RNA can be accomplished post-transcriptionally using vaccinia virus capping enzyme to generate a "Cap 0" structure: m7G(5')ppp(5')G (New England Biolabs, Ipswich, MA). Cap 1 structures can be generated using vaccinia virus capping enzyme and 2'-O methyltransferase: m7G(5')ppp(5')G-2'-O-methyl. The Cap 2 construct can be generated from the Cap 1 construct, followed by 2'-O-methylation of the 5'-antepenultimate nucleotide using a 2'-O methyltransferase. The Cap 3 construct can be generated from the Cap 2 construct, followed by 2'-O-methylation of the 5'-preantepenultimate nucleotide using a 2'-O methyltransferase. The enzyme can be derived from a recombinant source.

[0540] An mRNA molecule comprising at least one of the above-mentioned stabilizing elements can significantly increase the expression level of the protein it encodes, and when two or more of the above-mentioned stabilizing elements are used, it shows a synergistic effect in protein expression compared to mRNA using a single stabilizing element.

[0541] In some embodiments, the mRNAs described herein are depleted of one or more AU-rich sequences. AU-rich sequences, also known as AURES, are destabilizing sequences found in the 3' UTR.

[0542] In some embodiments, the mRNA described in the present disclosure further comprises an open reading frame (ORF) encoding a signal peptide. The signal peptide may comprise amino acids 15-60 at the N-terminus of the protein, which are typically required for transmembrane transport across the secretory pathway and, therefore, generally control the secretory pathway in which most proteins enter eukaryotes and prokaryotes. The length of the signal peptide may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 amino acids.

[0543] An "open reading frame" is a continuous stretch of DNA that begins with a start codon (eg, methionine (ATG)) and ends with a stop codon (eg, TAA, TAG, or TGA) and encodes a polypeptide.

[0544] Signal peptides from heterologous genes are known in the art and can be tested for desired properties and then incorporated into the nucleic acids of the present disclosure. In some embodiments, the signal peptide can include one of the following sequences: MDSKGSSQKGSRLLLLLVVSNLLLPQGVVG (SEQ ID NO: 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), and MFVFLVLLPLVSSQC (SEQ ID NO: 161).

[0545] The nucleic acid molecules provided herein can be synthesized by solid phase technology, liquid phase chemical synthesis, enzymatic ligation, and combinations thereof. The synthesized nucleic acid molecules can be purified and quantified. For details, see WO2021222304, the entire contents of which are incorporated herein by reference.

[0546] 7. Carrier

[0547] In another aspect, the present disclosure further provides a vector comprising a nucleic acid molecule as provided in the present disclosure, wherein the nucleic acid molecule is operably linked to at least one polynucleotide regulatory element (eg, a promoter) to express the chimeric protein construct encoded by the nucleic acid molecule.

[0548] In some embodiments, the vector is selected from a plasmid, a nanoplasmid, a cosmid, a viral vector, a minicircle, an RNA vector, or a linear or circular DNA (eg, transposon DNA) or RNA molecule.

[0549] In some embodiments, the viral vector is selected from a retrovirus, a lentiviral vector, an adenovirus, a parvovirus (e.g., adeno-associated virus), an adeno-associated virus (AAV) vector, a coronavirus, a negative-strand RNA virus such as an orthomyxovirus (e.g., influenza virus), a rhabdovirus (e.g., rabies and vesicular stomatitis virus), a paramyxovirus (e.g., malabsorption and sendai), a positive-strand RNA virus such as a picornavirus and an alphavirus, and a double-stranded DNA virus, including adenovirus, herpesvirus (e.g., herpes simplex virus type 1 and 2, Epstein-Barr virus, cytomegalovirus) and poxvirus (e.g., vaccinia virus, fowlpox virus and canarypox virus), a Norwalk virus, a togavirus, a flavivirus, a reovirus, a papovavirus, a hepadnavirus, a baculovirus and a hepatitis virus, a virus-like particle (VLP).

[0550] In some embodiments, the viral vector is a retroviral vector.

[0551] In some embodiments, the retrovirus is selected from the group consisting of avian leukocytoblastoma, mammalian C-type, B-type, D-type viruses, HTLV-BLV collection, lentivirus, and foamy virus.

[0552] In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is an oncolytic viral vector. An oncolytic viral vector refers to an oncolytic virus-based viral vector into which an exogenous nucleic acid sequence can be inserted.

[0553] In some embodiments, the lentiviral vector is selected from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or ovine demyelinating leukoencephalitis lentivirus.

[0554] In some embodiments, the carrier is an expression vector based on a transposon. A transposon is a DNA sequence that can change its position within the genome. In a transposon system, the nucleic acid molecule flanking the chimeric protein construct provided herein is flanked by terminal repeats identifiable by a transposase that mediates transposon movement. The transposase can be co-delivered as a protein, encoded on the same carrier as the chimeric protein construct, or encoded on an independent carrier. Non-limiting examples of transposon systems include Sleeping Beauty, Piggyback, Frog Prince, and Prince Charming.

[0555] In some embodiments, the vectors provided by the present disclosure can be combined with other vectors comprising other nucleic acid molecules encoding at least one co-expression moiety.

[0556] In some preferred embodiments, the vector further comprises a promoter; preferably, the promoter is an EF1α promoter or a CMV promoter.

[0557] In some preferred embodiments, the chimeric protein construct provided by the present disclosure can be expressed in the same vector and the same promoter as the co-expression part, or under different promoters, or in multiple vectors.

[0558] 8. Cells

[0559] In another aspect, the present disclosure further provides an engineered cell, which expresses the chimeric protein construct provided by the present disclosure, or comprises the chimeric nucleic acid construct or vector provided by the present disclosure.

[0560] In some embodiments, the engineered cells simultaneously express the chimeric protein construct as provided in the present disclosure and the coexpression portion as described above. The coexpression portion and the targeting protein binding domain of the chimeric protein construct can be combined to achieve a specific purpose. For example, when the targeting protein binding domain specifically identifies the protein that can cause immune rejection expressed on immune cells, the coexpression portion can be a protein that further suppresses immune rejection (for example, viral ER resident glycoproteins, etc.), a protein that targets the tumor microenvironment (for example, chemokine receptors, etc.), a protein that stimulates immune activity (for example, cytokines, immune cell germline proteins CD7, CD5, etc.), and the resulting cells will have reduced immunogenicity (or immune rejection) and / or have enhanced immune activity when transplanted into the human body. This is very useful for cellular immunotherapy and other therapeutic cell transplants. Therefore, the cells can be immune cells (for example, T cells), stem cells, kidney cells, pancreatic islet cells, and cardiomyocytes, etc.

[0561] In some embodiments, the cell is an immune cell selected from the group consisting of: T cells, natural killer (NK) cells, B cells, macrophages, monocytes, dendritic cells, neutrophils or γδT cells. The T cells can be selected from the group consisting of: CD8+T cells, CD4+T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, regulatory T cells, natural killer T cells, γ-δ T cells, cytokine-induced killer (CIK) T cells and tumor infiltrating lymphocytes.

[0562] In some embodiments, the engineered cells are T cells. In the case of expressing the chimeric protein constructs and co-expression moieties described above to obtain T cells with reduced immunogenicity and / or enhanced immune activity, the T cells may further express receptors that can specifically target tumor-associated markers, including but not limited to CAR, TCR, NK cell activating receptor components, etc.

[0563] In some embodiments, the engineered cells are T cells that simultaneously express a chimeric protein construct as provided herein and one or more selected from the group consisting of adenovirus E3-K19, CAR, NK cell activating receptor, and CNK signaling transduction component.

[0564] In some embodiments, the engineered cells are T cells that simultaneously express a chimeric protein construct as provided herein and a NK cell activating receptor and optionally a CNK signaling transduction component.

[0565] In some embodiments, the engineered cell is a T cell that simultaneously expresses a chimeric protein construct as provided in the present disclosure, adenovirus E3-K19, and CAR.

[0566] In another aspect, the present disclosure also provides a method for producing the engineered cells described herein (e.g., T cells), comprising introducing the vector described herein into a starting cell under conditions suitable for expressing the nucleic acid molecule described herein.

[0567] Many methods for producing CAR-T cells known in the art can also be applied to produce engineered cells as described herein. For example, Zhang et al., Engineering CAR-T cells, Biomarker Research (2017) 5: 22 describe methods for producing CAR-T cells. The methods provided herein may include one or more steps selected from the following: obtaining starting cells, culturing (including amplification, optionally including activation) starting cells, and genetically modifying cells. The starting cells can be stem cells, which can be hematopoietic progenitor cells (e.g., T cell progenitors, NK cell progenitors, macrophage progenitors), hematopoietic stem cells (HSC), CD34+ cells, embryonic cell line stem cells, mesenchymal stem cells, or iPSC cells. The starting cells can also be cells differentiated from stem cells, such as the immune cells described above.

[0568] The starting cells can be obtained from any source, for example, immune cells (e.g., T cells) that can be separated from a subject (e.g., a human subject). In some embodiments, immune cells are obtained from a subject of interest, such as a subject suspected of having a specific disease or condition, a subject suspected of having a susceptibility to a specific disease or condition, a subject who will experience, is experiencing, or has received treatment for a specific disease or condition. The subject can also be a healthy volunteer or a healthy donor; immune cells can also come from a blood bank. The immune cells can be autologous or allogeneic for the subject of interest. The immune cells can be collected from any location in the subject where they are present, including but not limited to blood, umbilical cord blood, spleen, thymus, lymph nodes, pleural effusion, splenic tissue, tumors, and bone marrow. The separated immune cells can be used directly or stored for a period of time, such as freezing.

[0569] The activation and / or expansion of immune cells is one of the main steps of immune cell function. In some embodiments, immune cells are activated and expanded while, before or / and after genetic modification. In some embodiments, immune cells are activated and / or expanded in vitro, in vitro or in vivo. The method of activating and expanding immune cells has been described in the art and can be used in the method described herein. For example, T cells can be activated and expanded by contacting with the surface of the ligand of the co-stimulatory molecule on the surface of the medicament and the stimulating CD3 / TCR complex related signal attached. In particular, T cell colonies can be stimulated, such as by contacting with anti-CD3 antibodies or their antigen-binding fragments or anti-CD2 antibodies fixed on the surface, or contacting with protein kinase C activators (such as bryostatin) and calcium ion carriers. In order to costimulate the auxiliary molecules on the surface of T cells, a ligand combined with the auxiliary molecule is used. For example, under conditions suitable for stimulating T cell proliferation, T cell colonies can be contacted with anti-CD3 antibodies and anti-CD28 antibodies. In order to stimulate the proliferation of CD4+T cells or CD8+T cells, anti-CD3 antibodies and anti-CD28 antibodies can be used. In certain embodiments, the primary stimulation signal and the co-stimulatory signal for T cells can be provided by different schemes.

[0570] Genetically modified cells can be achieved by transducing substantially homogeneous starting cells with nucleic acid molecules encoding chimeric protein constructs provided herein. In certain embodiments, nucleic acid molecules provided herein are introduced into starting cells using retroviral vectors (e.g., slow virus vectors). For example, nucleic acid molecules provided herein can be cloned into slow virus vectors, and expression can be driven from their endogenous promoters, slow virus long terminal repeats, or promoters specific to target cell types of interest. Common delivery methods for delivering viral vectors include, but are not limited to, electroporation, microinjection, gene guns, and magnetic transfection.

[0571] Genetically modified cells can also be achieved by delivering nucleic acid molecules with LNP, as specifically described in the section "6. Nucleic Acid Molecules" above. Other non-viral methods for nucleic acid delivery include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALE nucleases, CRISPR) can also be used to achieve genetic modification of the starting cells, thereby obtaining engineered cells as described in the present disclosure.

[0572] In certain embodiments, provided herein is an engineered cell by transfecting the nucleic acid molecules encoding the chimeric protein construct provided herein into starting cells before administration. In certain embodiments, provided herein is an engineered cell by being prepared by, for example, viral vector transfection immune cells with the nucleic acid molecules encoding the chimeric protein construct provided herein. Provided herein is an engineered cell showing reduced immunogenic molecules (for example, TCR, HLA, etc.) expression and / or immunosuppressive molecules (for example, PD-1, etc.) expression and / or enhanced tumor-targeted receptors (for example, CAR, engineered TCR, CNK receptors, etc.) expression on the cell surface.

[0573] On the other hand, the present disclosure also provides cell populations produced in vitro by the above methods. In certain embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the cell population express a detectable level of expression marker polypeptide provided herein (e.g., an EGFR truncate (EGFRt) that can be cleaved and connected to a chimeric protein construct as described herein). The expression level of the EGFR truncate can represent the expression level of the chimeric protein construct as described herein in the cell population.

[0574] 9. Pharmaceutical compositions and kits

[0575] In another aspect, the present disclosure further provides a pharmaceutical composition or a kit comprising (i) a chimeric protein construct, nucleic acid molecule, vector, or cell population as described herein, and (ii) a pharmaceutically acceptable vehicle. The term "pharmaceutically acceptable vehicle" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that can facilitate the storage and administration of the chimeric protein construct, nucleic acid molecule, expression vector, and / or cell population provided herein. Pharmaceutically acceptable vehicles can include any suitable component, such as, but not limited to, saline, liposomes, polymeric excipients, colloids, or carrier particles.

[0576] In certain embodiments, the pharmaceutically acceptable medium is a saline solution that can dissolve or disperse the chimeric protein constructs, nucleic acid molecules, expression vectors, and / or cell populations provided herein. Illustrative examples of saline solutions include, but are not limited to, buffered saline, sucrose solutions, physiological saline, acetate buffer, phosphate buffer, citrate buffer, bicarbonate buffer, saline solution, and polysorbate solution.

[0577] i) Nucleic acid pharmaceutical compositions

[0578] In certain embodiments, pharmaceutically acceptable medium is liposome.Liposome is unilamellar or multilamellar vesicle, has the film and internal aqueous part formed by lipophilic material.The nucleic acid molecule and / or carrier provided by the present disclosure can be encapsulated in the aqueous part of liposome.Exemplary liposome includes but is not limited to liposome based on 3 [N- (N ', N '-dimethylaminoethane) aminoformyl] cholesterol (DC-Chlo), based on N- (2,3- dioleoyloxy) propyl group-N, N, N- trimethylammonium chloride (DOTMA) liposome, and based on 1,2- dioleoyloxy -3- trimethylpropane (DOTAP) liposome.Prepare liposome and the method that nucleic acid molecule and / or carrier are encapsulated in liposome is well known in the art (referring to, for example, DDLasic et al, Liposomes in gene delivery, published by CRC Press, 1997).

[0579] In certain embodiments, the pharmaceutically acceptable medium is a polymeric excipient, including but not limited to microspheres, microcapsules, polymeric micelles and dendrimers. The nucleic acid molecules and / or vectors provided herein can be encapsulated, adhered or coated on a polymer-based component by methods known in the art (see, for example, W. Heiser, Nonviral gene transfer techniques, published by Humana Press, 2004; US patent 6025337; Advanced Drug Delivery Reviews, 57(15):2177-2202 (2005)).

[0580] In certain embodiments, the pharmaceutically acceptable medium is a colloid or carrier particle, such as gold colloid, gold nanoparticles, silica nanoparticles and multi-segment nanorods. The nucleic acid molecules and / or carriers provided herein can be coated, adhered to or combined with a carrier in any suitable manner known in the art (see, for example, M. Sullivan et al., Gene Therapy, 10: 1882–1890 (2003), C. McIntosh et al., J. Am. Chem. Soc., 123(31): 7626–7629 (2001), D. Luo et al., Nature Biotechnology, 18: 893-895 (2000), and A. Salem et al., Nature Materials, 2: 668-671 (2003)).

[0581] In certain embodiments, the pharmaceutical composition may further comprise additives, including but not limited to stabilizers, preservatives, and transfection facilitators that facilitate cellular uptake of the drug. Suitable stabilizers may include but are not limited to sodium glutamate, glycine, EDTA, and albumin. Suitable preservatives may include but are not limited to 2-phenoxyethanol, sodium benzoate, potassium sorbate, methyl hydroxybenzoate, phenols, thimerosal, and antibiotics. Suitable transfection facilitators may include but are not limited to calcium ions.

[0582] The pharmaceutical compositions provided herein can be administered by any suitable route known in the art, including but not limited to, parenteral, oral, enteral, buccal, nasal, topical, rectal, vaginal, intramuscular, intranasal, transmucosal, epidermal, transdermal, dermal, ocular, pulmonary and subcutaneous routes of administration. The pharmaceutical compositions provided herein can be administered to a subject in the form of a formulation or formulation suitable for each route of administration. Preparations suitable for pharmaceutical composition administration can include but not limited to solutions, dispersions, emulsions, powders, suspensions, aerosols, sprays, nasal drops, liposome-based formulations, patches, implants and suppositories.

[0583] The formulations can be conveniently presented in unit dosage form and can be prepared by any method well known in the pharmaceutical art. The method of preparing these formulations or pharmaceutical compositions comprises the step of providing the nucleic acid molecules described herein to one or more pharmaceutically acceptable vehicles and optionally one or more additives. Methods for preparing such formulations can be found in, for example, Remington's Pharmaceutical Sciences (Remington: The Science and Practice of Pharmacy, 19th ed., A. R. Gennaro (ed), Mack Publishing Co., NJ, 1995; R. Stribling et al., Proc. Natl. Acad. Sci. USA, 89: 11277-11281 (1992); A. Barnes et al., Current Opinion in Molecular Therapeutics 2000 2: 87-93 (2000); T. W. Kim et al., The Journal of Gene Medicine, 7(6): 749-758 (2005); and S. F. Jia et al., Clinical Cancer Research, 9: 3462 (2003); A. Shahiwala et al., Recent patents on drug delivery and formulation, 1: 1-9 (2007);, which reference is incorporated herein by reference in its entirety.

[0584] In some embodiments, the nucleic acid molecule (eg, mRNA) can be delivered physically, biologically, or chemically (see, e.g., S. Guan, J. Rosenecker, Gene Ther. 2017, 24, 133.).

[0585] Physical methods include, but are not limited to, delivery by gene gun (e.g., a gene gun using Au-particles), electroporation, sonoporation, and the like (see, e.g., Kutzler et al., (2008) DNA vaccines: Ready for prime time? Nat Rev Genet 9:776–788; Geall et al., Nonviral delivery of self-amplifying RNA vaccines. Proc Natl Acad Sci US A. Sep. 4, 2012; 109(36):14604-9.).

[0586] Biological methods include, but are not limited to, delivery via viral vectors (eg, retroviral vectors, adenoviral vectors, adeno-associated viral vectors).

[0587] Chemical methods include, but are not limited to, delivery via natural proteins / polysaccharides, polymers, and lipids. Exemplary natural proteins / polysaccharides include protamine and chitosan (see, e.g., AE et al., Cancer Immunol. Immunother. 2015, 64, 1461; US ​​Kumar et al. ACS Nano 2021, 11, 17582). Exemplary polymers include polyethyleneimine (PEI) (e.g., linear PEI, branched PEI, and dendritic PEI), poly (β-amino ester) (PBAE) (see, for example, K. Singha et al., Nucleic Acid Ther. 2011, 21, 133; AA Eltoukhy et al., Biomaterials 2012, 33, 3594).

[0588] Exemplary lipids include cationic lipids such as 1,2-diocta-decenyl-3-trimethy lammonium-propane (DOTMA) and 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) (see, e.g., X. Hou et al., Nat. Rev. Mater. 2021, 10, 1078.), liposomes formed by DOTMA, DOTAP and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, helper lipid), which can form colloid-stabilized nanoparticles after self-assembly with mRNA (see, e.g., L. M. Kranz et al., Nature 2016, 534, 396.).

[0589] Exemplary lipids can also be ionizable lipids. Ionizable lipids (pKa 6.5-6.9) are alternative lipid materials, neutral at physiological pH, but positively charged by the protonation of free amines in an acidic environment (see, for example, SC Semple et al., Nat. Biotechnol. 2010, 28, 172). After cell internalization, the nanoparticles formed by ionizable lipids are encapsulated in endosomes (endosomes). Subsequently, due to the continuous decline of pH value in endosomes and lysosomes, ionizable lipids obtain protons for ionization, thereby promoting the fusion of lipid nanoparticles (lipid nanoparticle, LNP) with endosomal membranes, ultimately causing the mRNA loaded on lipid nanoparticles to be released into cytoplasm (see, for example, L. Miao et al., Mol. Cancer 2021, 20, 41.).

[0590] Ionizable lipids can be combined with cholesterol, helper lipids, and PEGylated lipids (i.e., PEGylated lipids) to form lipid nanoparticle formulations. Cholesterol is a naturally rigid and hydrophobic lipid that maintains the structure and stability of lipid nanoparticles. It can also promote the fusion of mRNA-loaded lipid nanoparticles (i.e., mRNA nanoparticles) with endosomal membranes. Helper lipids (such as zwitterionic lipids DOPE, 1,2-distearoyl-snglycero-3-phosphocholine (DSPC) and 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)) are widely used to promote cell membrane penetration and endosomal membrane escape (see, for example, N. Chaudhary et al., Nat. Rev. Drug Discovery 2021, 20, 817.). PEGylated lipids are composed of PEG and anchor lipids. The hydrophilic PEG is mainly distributed on the surface of the mRNA complex, while the hydrophobic region is embedded in the interior of the lipid bilayer. The introduction of PEGylated lipids not only increases the half-life of lipid nanoparticles but also can regulate the particle size by changing the molecular weight of the PEG chain. Typically, the molecular weight and lipid tail length can range from 350 to 3000 Da and 10 to 18 carbons, respectively (see, for example, N. Chaudhary et al., Nat. Rev. Drug Discovery 2021, 20, 817.).

[0591] Over the past few decades, researchers have developed a large library of ionizable lipids for mRNA delivery, including DLin-MC3-DMA, SM-102, TT3, C12-200, 306O i10and ALC-0315 (see, for example, M. Yanez Arteta et al., Proc. Natl. Acad. Sci. USA 2018, 115, E3351; R. Verbeke et al., Controlled Release 2021, 333, 511; B. Li et al., Nano Lett. 2015, 15, 8099; KA Hajj et al., Nano Lett. 2020, 20, 5167; KA Hajj et al., Small 2019, 15, 1805097; AB Vogel et al., Nature 2021, 592, 283). Some of them have achieved remarkable results in clinical applications. A typical example is DLin-MC3-DMA, which is a key component of Onpattro approved by the U.S. Food and Drug Administration (FDA) for siRNA delivery (see, for example, A.Akinc et al., Nat.Nanotechnol.2019,14,1084). DLin-MC3-DMA is also widely used in mRNA delivery, including protein and peptide replacement, gene editing and antiviral infection (see, for example, RSRiley et al., Sci.Adv.2021,7,eaba1028.). SM-102 and ALC-0315, two "star molecules", have been approved by the FDA as key components in BNT162b and mRNA-1273 vaccines for preventing COVID-19, respectively (see, for example, X.Hou et al., Nat.Rev.Mater.2021,10,1078.).

[0592] An ideal lipid-based mRNA carrier must meet the following conditions: 1) naked mRNA can form a stable complex to protect it from degradation; 2) four key components (ionizable lipids, cholesterol, helper lipids, and PEGylated lipids) should be added to stabilize the mRNA complex; 3) the components of the lipid nanoparticles should be protonated to induce membrane destabilization and promote endosomal escape of the mRNA complex; and 4) all lipid materials are biodegradable and do not cause any harm to patients.

[0593] The following points should be considered when optimizing lipid-based delivery platforms: 1) Degradability of ionizable lipids: The backbone structure of lipids promotes lipid clearance and reduces toxicity by introducing alkyne and ester groups into the lipid tail; 2) Immunogenicity of lipid nanoparticles: Heterocyclic lipids within lipid nanoparticles can improve the efficiency of mRNA vaccines by activating the interferon gene stimulator (STING) pathway of dendritic cells (DCs) (see, for example, L. Miao et al., Nat. Biotechnol. 2019, 37, 1174.); 3) Stability of lipid nanoparticles: Some promising strategies are expected to improve the stability of mRNA vaccines, including pK a optimization, excipient introduction and mRNA modification, etc.

[0594] Lipid nanoparticles can be produced using components, compositions, and methods as are known in the art, see, for example, PCT / US2016 / 052352, PCT / US2016 / 068300, PCT / US2017 / 037551, PCT / US2015 / 027400, PCT / US2016 / 047406, PCT / US2016 / 000129, PCT / US2016 / 01428 ...7 / 0 6 / 014280, PCT / US2017 / 038426, PCT / US2014 / 027077, PCT / US2014 / 055394, PCT / US2016 / 052117, PCT / US2012 / 069610, PCT / US2017 / 027492, PCT / US2016 / 059575, and PCT / US2016 / 069491, the entire contents of which are incorporated herein by reference.

[0595] ii) Oncolytic viruses and pharmaceutical compositions thereof

[0596] In some embodiments, the present application provides an oncolytic virus capable of expressing the chimeric protein construct described herein. In this application, "oncolytic virus" refers to any virus that can infect tumor cells, replicate in tumor cells, and dissolve tumor cells. In certain embodiments, the oncolytic virus is further capable of spreading to other tumor cells in a continuous replication cycle.

[0597] Oncolytic viruses can be derived from a variety of viruses, non-limiting examples of which include vaccinia virus, adenovirus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), myxoma virus, reovirus, poliovirus, vesicular stomatitis virus (VSV), measles virus (MV), Lassa virus (LASV) and Newcastle disease virus (NDV), as well as variants of these viruses.

[0598] In some embodiments, the present application provides an oncolytic virus comprising a nucleic acid molecule as provided herein, which is operably linked to at least one polynucleotide regulatory element (e.g., a promoter) to express a chimeric protein construct encoded by the nucleic acid molecule.

[0599] In some embodiments, the oncolytic viruses described herein can infect substantially any cell type. In some embodiments, the oncolytic viruses can be replication selective. Replication selective oncolytic viruses replicate more in tumor cells than in non-tumor cells.

[0600] It is understood that oncolytic viruses can be made replication selective if viral replication is under the control of gene expression regulators, such as the enhancer / promoter region derived from the 5' side of the albumin gene (see, for example, Miyatake et al., 1997, J. Virol. 71: 5124-5132).

[0601] In some embodiments, the oncolytic virus may have a tumor cell-specific promoter or a tumor cell-specific transcriptional regulatory sequence in its genome. "Tumor cell specificity" for a promoter or transcriptional regulatory sequence means that it is typically present in target tumor cells at a higher level than normal cells. In this way, an enhanced level of tumor cell specificity can be conferred oncolytic viruses.

[0602] For example, by operably linking the HSV gene to the TGF-β promoter, the main transcription unit of HSV can be placed under the transcriptional control of the tumor growth factor-β (TGF-β) promoter. It is known that certain tumor cells overexpress TGF-β relative to non-tumor cells of the same type. Therefore, an oncolytic virus in which replication is subject to the transcriptional control of the TGF-β promoter is replication selective because it is more capable of replicating in certain tumor cells than in non-tumor cells of the same type. Similar replication selective oncolytic viruses can be prepared using any gene expression regulator known to selectively cause overexpression in affected cells. Replication selective oncolytic viruses can be, for example, HSV-1 mutants in which the gene encoding ICP34.5 is mutated or deleted. Oncolytic viruses can also further include other modifications in their genome. For example, an oncolytic virus can include additional DNA inserted into the UL44 gene. This insertion can produce functional inactivation of the UL44 gene and the resulting lysis phenotype, or it can be inserted into an already inactivated gene, or replace a deleted gene.

[0603] 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.

[0604] 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.

[0605] In some embodiments, the oncolytic virus vector can express the chimeric protein construct described in the present application, and the targeting domain of the chimeric protein construct can recognize and bind to tumor proteins. Any tumor-related protein or tumor-related target described above in this application is applicable. In certain embodiments, the tumor protein can be a protein highly expressed in tumor cells (e.g., c-Myc, Bcl-2, Bcl-xL, Bcl-w, KRAS), a protein expressed in tumor cells that promotes tumor cell growth (e.g., estrogen receptor, androgen receptor, Her2, VEGF, VEGFR, PDGFRβ, EGFR, EGFR mutants), or a protein that promotes tumor cells to escape normal immune responses (e.g., PD-L1, TGF-β1).

[0606] In some embodiments, the targeting domain of the chimeric protein construct can bind to Bcl-2. Any targeting domain capable of targeting Bcl-2 can be used, for example, but not limited to, the development of nanoantibody sequences targeting the highly conserved and homologous BH1, BH2 motifs of Bcl-2 anti-apoptotic family members can be used as targeting domains. After the ER-TPD element targeting Bcl-2 family proteins is constructed and cloned and loaded onto a strong oncolytic virus promoter, after specific infection of tumor cells, the oncolytic virus is amplified in large quantities and expresses the ER-TPD element of the Bcl-2 family protein, thereby achieving degradation of the intracellular Bcl-2 anti-apoptotic member proteins and directly leading to tumor cell apoptosis.

[0607] In some embodiments, the targeting domain of the chimeric protein construct can bind to VEGFR. Any targeting domain capable of targeting VEGFR can be used, for example, but not limited to, VEGFR antibodies (anti-VEGF-A antibody Bevacizumab and anti-VEGFR2 antibody Ramucizumab,) heavy chain and light chain recombinant scFv series ER-TPD element, constructing a targeting VEGFR ER-TPD element, and cloning and loading it on a strong oncolytic virus promoter. After specific infection of tumor cells, the oncolytic virus is amplified in large quantities and expresses the VEGFR ER-TPD element, directly inhibiting the expression of VEGFR in tumor cells, resulting in growth inhibition and apoptosis of tumor cells due to lack of sufficient nutrients.

[0608] In some embodiments, the targeting domain of the chimeric protein construct can bind to TGF-β1 and PD-L1. Any targeting domain that can target and bind to bcl-2 can be used, for example, but not limited to, using the heavy chain and light chain recombinant scFv of TGF-β1 and PD-L1 antibodies in series with ER-TPD elements to construct TGF-β1 and PD-L1ER-TPD elements, and cloning and loading them on the strong promoter of oncolytic virus. After specific infection of tumor cells, the oncolytic virus is amplified in large quantities and expresses TGF-β1 and PD-L1ER-TPD elements, directly inhibiting and degrading the expression of TGF-β1 and PD-L1 in tumor cells, breaking the tumor immunosuppressive microenvironment, and thus achieving the effect of treating tumors.

[0609] Without being limited by theory, it is believed that the chimeric protein construct of the present application has unique advantages when expressed in tumor cells via an oncolytic viral vector. On the one hand, the chimeric protein construct of the present application can target and degrade proteins in tumor cells that help tumor cells grow or escape immune system or resist apoptosis, thereby promoting apoptosis or death of tumor cells. On the other hand, the oncolytic viral vector can specifically replicate in tumor cells, promoting tumor cell lysis through viral replication and lysis. By integrating two different tumor-killing methods into the oncolytic viral vector, a synergistic killing effect can be achieved in the same drug form.

[0610] In another aspect, the present disclosure further provides a pharmaceutical composition comprising an oncolytic virus provided herein and a pharmaceutically acceptable medium. In certain embodiments, the pharmaceutically acceptable medium comprises any and all transport vehicles, solvents, diluents, excipients, adjuvants, dispersion media, coatings, antibacterial and antifungal agents, absorbents, and the like that are compatible with administration in mammals, and particularly human subjects.

[0611] In certain embodiments, the oncolytic virus or its pharmaceutical composition described herein is formulated for intravenous or intratumoral administration. In certain embodiments, the oncolytic virus or its pharmaceutical composition may be placed in a solvent or diluent suitable for human or animal use. The solvent or diluent is preferably isotonic, hypotonic or weakly hypotonic and has a relatively low ionic strength. Typical examples include sterile water, normal saline (such as sodium chloride), Ringer's solution, glucose, trehalose or sucrose solution, Hank's solution and other aqueous physiologically balanced salt solutions.

[0612] In one embodiment, the oncolytic virus or its pharmaceutical composition described herein may have a buffer. Suitable buffers include, but are not limited to, phosphate buffer (e.g., PBS), bicarbonate buffer, and / or Tris buffer capable of maintaining a physiological or slightly alkaline pH (e.g., from about pH 7 to about pH 9).

[0613] In one embodiment, the oncolytic virus composition of the present invention may be prepared to improve its stability, especially under production conditions and for long-term (ie, at least 6 months, preferably at least 2 years) frozen (eg, -70 ° C, -20 ° C) storage, refrigerated storage (eg, 4 ° C), and room temperature storage. A variety of existing viral preparations in the art are in either frozen liquid form or in freeze-dried form (eg, WO98 / 02522 and WO2008 / 114021, etc.). A solid (eg, dry powder or freeze-dried) composition can be obtained by steps involving vacuum drying and freeze-drying. For illustrative purposes, a buffered formulation with the addition of NaCl and sugar is particularly suitable for preserving viruses.

[0614] iii) Cellular pharmaceutical compositions

[0615] In another aspect, the present disclosure also provides a cell pharmaceutical composition comprising the engineered cells or cell populations provided by the present disclosure and a pharmaceutically acceptable medium. Exemplary media include buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In one aspect, the cell pharmaceutical composition of the present invention is formulated for intravenous administration.

[0616] 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).

[0617] In certain embodiments, the cells to be transplanted are immune cells (e.g., T cells), stem cells (and cells differentiated therefrom), kidney cells, pancreatic islet cells, cardiomyocytes, etc. In certain embodiments, the cells to be transplanted can be autologous cells or allogeneic cells.

[0618] On the one hand, the application also provides a cell to be transplanted that expresses the chimeric protein construct, nucleic acid molecule or vector described above, wherein the chimeric protein construct described in the application is expressed, and the chimeric protein construct comprises an ERAD mechanism protein binding domain and a targeting domain, and the targeting domain comprises a domain that specifically targets or binds to a target protein, and the target protein is a transplant rejection-related protein. In certain embodiments, the chimeric protein construct further comprises a protein degradation pathway member (e.g., E3 ubiquitin ligase, proteasome, lysosome) binding domain as described in the application. Any protein degradation pathway member binding domain described in the application can be used.

[0619] In certain embodiments, the transplant rejection reaction-related proteins include antigen presenting molecules (such as MHC class I molecules, MHC class II molecules, MICA / B molecules, etc.), antigen recognition molecules (such as TCR, CD123, NKG2D, etc.), immune checkpoint molecules (such as PD-1, PD-L1, CTLA4, TIM3, TIGIT, LAG3, A2AR, BTLA, IDO1, IDO2, TDO, KIR, NOX2, VISTA, SIGLEC7, PVR, etc.), etc.

[0620] In certain embodiments, the transplant rejection-related protein comprises an immunogenic protein (eg, HLA (HLAα / β), TCR (αβTCR), NKG2D (natural killer cell group 2 member D) ligand, etc.).

[0621] The present disclosure also provides the above-mentioned cells to be transplanted and treatment methods thereof, which can overcome the problems faced in existing cell transplantation and cell therapy, such as the source of personalized transplanted cells and therapeutic cells, the inability to standardize and scale production, and low treatment efficiency.

[0622] The immune system has powerful plasticity and a nearly unlimited ability to detect invading viruses, bacteria, foreign cells, and diseased cells. This extraordinary immune surveillance capability is primarily achieved through humoral and cellular immunity, which include two important molecular structures: immunoglobulins and T cell receptors (TCRs). The TCR is the defining structure of T cells and is a transmembrane heterodimer composed of α and β chains or δ and γ chains connected by disulfide bonds. Within these chains, the complementarity determining regions (CDRs) determine the antigens that the TCR will bind to. Within the TCR, the TCRα and TCRβ subunits (or TCRγ and TCRδ in γδ T cells) are responsible for recognizing major histocompatibility complex (MHC) / antigen ligands.

[0623] The human MHC class I gene region includes alleles at the HLA-A, B, and C loci, encoding classic class I antigens (molecules), such as HLA-A, B, and C antigens, known as HLA-A, HLA-B, and HLA-C. These antigen molecules are present on the surface of all somatic cells and bind to intracellular protein epitope peptides for recognition by the immune system. If a cell produces a mutant protein or is invaded by foreign bacteria or viruses, the cell presents these mutant proteins or heterologous protein epitopes. Immune cells recognize these mutant proteins or heterologous protein epitopes and initiate an immune attack and killing, thereby eliminating the diseased cells, pathogens, and viruses that invade the cells.

[0624] α / β T lymphocytes recognize peptide-MHC ligands through a multimeric protein assembly called the αβ T cell antigen receptor (TCR) CD3 complex. This structure consists of a variable αβ TCR dimer that binds antigen and three invariant dimers (CD3γε, δε, and ζζ) that participate in TCR.CD3 surface trafficking, stabilization, and signal transduction. The αβ T cell receptor (αβ TCR) is expressed on the majority (approximately 95%) of T cells and plays a key role in T cell activation by recognizing major histocompatibility complex (MHC)-anchored antigens. Therefore, TCR-mediated T cell activation is a key step in the pathogenesis of graft-versus-host disease (GVHD) during allogeneic hematopoietic cell transplantation (allo-HCT) and allogeneic CAR-T cell therapy.

[0625] The human leukocyte antigen (HLA) system or complex is a group of related proteins encoded by the human major histocompatibility complex (MHC) gene complex. These cell-surface proteins are responsible for regulating the immune system. In the therapeutic transplantation setting, "HLA mismatch" occurs when the donor HLA on the allograft differs from that of the recipient. HLA mismatch leads to the activation of alloreactive T cells, which can cause acute cellular rejection (ACR) within six months of transplantation. Mismatched donor HLA antigens are also targets for the development of de novo donor-specific HLA antibodies (dnDSA), which play a reinforcing role in acute and chronic rejection of transplanted cells (such as T cells). Therefore, to generate universal transplantable cells (such as T cells) for safe allogeneic infusion and therapeutic purposes, effective blockade of graft-versus-host disease (GVHD) by genetically disrupting the TCR is recommended. In addition, it is necessary to suppress HLA expression on allogeneic cells (such as T cells) to reduce the recipient's immune system's rejection of the allogeneic T cell TCRαβ and / or HLA class I.

[0626] In certain embodiments, the cells to be transplanted that express the chimeric protein constructs, nucleic acid molecules, or vectors described above have reduced immunogenicity.

[0627] In some embodiments, the cells to be transplanted with reduced immunogenicity (or with high compatibility) described in the present application are T cells, which express a TPD chimeric protein construct comprising an scFv that specifically recognizes TCR and / or HLA and a transmembrane domain or a functional variant thereof and an endoplasmic reticulum retention domain or a functional variant thereof of the HCMV glycoprotein US2 and / or US11.

[0628] The ERAD machinery protein binding domain comprises an amino acid sequence as shown in SEQ ID NO.112 or SEQ ID NO.118, 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.

[0629] In some embodiments, the cells to be transplanted with reduced immunogenicity are T cells, which express an ER-TPD chimeric protein construct comprising a scFv that specifically recognizes TCR and a transmembrane domain of adenovirus E3-K19 or a functional variant thereof and an endoplasmic reticulum retention domain or a functional variant thereof. In some embodiments, the cells to be transplanted with reduced immunogenicity are T cells, which express an ER-TPD chimeric protein construct comprising a targeting protein binding domain and an ERAD mechanism protein binding domain, wherein the targeting protein binding domain comprises 6 CDRs comprising an amino acid sequence as shown in SEQ ID NO: 152 or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identity thereto. The ERAD mechanism protein binding domain comprises an amino acid sequence as shown in SEQ ID NO. 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.

[0630] In some embodiments, the above-mentioned T cell-expressed ER-TPD chimeric protein construct with reduced immunogenicity further comprises a protein degradation pathway member (e.g., E3 ubiquitin ligase, proteasome) binding domain, optionally, the protein degradation pathway member binding domain is connected to the ERAD mechanism protein binding domain. In some embodiments, the above-mentioned T cell-expressed ER-TPD chimeric protein construct with reduced immunogenicity further comprises an E3 ubiquitin ligase binding domain having an amino acid sequence as shown in SEQ ID NO: 162. In some embodiments, the above-mentioned T cell-expressed ER-TPD chimeric protein construct with reduced immunogenicity further comprises an E3 ubiquitin ligase binding domain having an amino acid sequence as shown in SEQ ID NO: 166.

[0631] In some embodiments, the cells to be transplanted (such as T cells) with reduced immunogenicity further express the co-expression moiety as described above.

[0632] For example, in order to reduce the immunogenicity or antigen presentation of cells, proteins that can degrade or reduce MHC class I or class II molecules can be expressed as co-expressed parts in the above-mentioned cells to be transplanted (such as T cells) with reduced immunogenicity (for example, complete viral ER-resident glycoproteins, including but not limited to HCMV US2, US3, US11, US10, adenovirus E3-K19, HCMV US6 and HSV ICP47). Effective downregulation of TCR will greatly inhibit TCR-mediated immune attack and reduce GVHD during allogeneic T cell infusion. Glycoproteins including natural viral ER-resident can further inhibit MHC molecules, thereby preventing peptides from being presented to recipient CD8+ T cells and inhibiting the immune recognition of allogeneic T cells. Therefore, the highly compatible T cells described above are also called "autologous universal T cells (UT)" or "universal T cells (UT)", which can achieve the purpose of allogeneic transfusion therapy and can improve the compatibility of allogeneic T cells and the long-term persistence after infusion.

[0633] On the other hand, the present disclosure provides a method for reducing the subject's immune response to cell transplantation, comprising administering to the subject a therapeutically effective amount of the cells to be transplanted as described above with reduced immunogenicity, thereby reducing the subject's immune response to the transplant or to the modified cells. In certain embodiments, the cells to be transplanted express a TPD chimeric protein construct as described herein in the cell, the TPD chimeric protein construct comprising a domain targeting immunogenic protein (e.g., HLA (HLA α / β), TCR (αβTCR), NKG2D (natural killer cell 2 family member D) ligand, etc.) and the ERAD mechanism protein binding domain described above, and optionally, a protein degradation pathway member binding domain.

[0634] 1. Cell therapy

[0635] Cell therapy involves genetically engineering cells (such as immune cells) to present tumor-associated antigens or express receptors that specifically recognize diseased cells. These cells are then exponentially expanded in vitro and then infused back into the patient's body, activating the immune system to attack tumor cells or directly and specifically recognize and kill them. In 2012, CD19-targeted CAR-T cells achieved the first-ever targeted elimination of tumor cells in a B-cell leukemia patient, becoming the next truly curative leukemia treatment after bone marrow stem cell transplantation and ushering in a new era of precision medicine cell therapy. This technology has the potential to be applied to the treatment of various hematologic malignancies and solid tumors. However, the current clinical efficacy of conventional CAR-T in treating solid tumors is poor. The reasons are: (1) The killing function of CAR-T is highly dependent on the recognition of tumor-associated antigens (TAA) by the CAR structure, but due to the heterogeneity of solid tumors, there are great differences in the expression of target proteins on the surface of tumor cells; currently, single-target CAR-T cannot completely recognize and kill all tumor cells, which leads to tumor immune escape and recurrence and metastasis; (2) The direct immunosuppressive effect of solid tumors, expressing PD-L1, B3H7, etc., directly inhibits the activation of T cells; (3) The immunosuppressive microenvironment of solid tumors contains a large number of immunosuppressive cells, such as TAM, Treg, and MDSC. It is difficult for conventional CAR-T to break through the limitations of immunosuppressive cells inside the tumor; (4) Conventional CAR-T is a personalized cell therapy. The T cells come from the patient himself. If the patient has received a lot of radiotherapy and chemotherapy, and the immune system function is damaged, it is difficult to isolate a sufficient number of T cells from the patient's peripheral blood. Even if they are isolated and transformed, the proliferation and killing function of T cells are still very weak, so it is difficult to achieve a therapeutic effect.

[0636] The cells to be transplanted (eg, T cells) provided in this application can overcome the problems faced in existing cell therapy, such as differences in tumor targets, tumor immunosuppressive environment, and sources of personalized therapeutic cells.

[0637] For the purpose of cell therapy, the cells to be transplanted (e.g., T cells, further, universal T cells with defects in TCRαβ and MHC molecule surface expression) with reduced immunogenicity can be further genetically engineered, for example, to express the co-expression portion as described above, to achieve therapeutic purposes.

[0638] For example, in order to enable the cells to be transplanted (such as T cells) to specifically act on disease-related target proteins or cells expressing disease-related target proteins, binding domains or molecules that recognize or bind to disease-related target proteins can be expressed in the above-mentioned cells to be transplanted (such as T cells) with reduced immunogenicity, such as chimeric antigen receptors CAR, engineered TCR, or CNK receptors provided in this application, or antiviral protein binding domains, etc.

[0639] In some embodiments, molecules (such as chemokine receptors or cytokine receptors) that can mediate the migration of the transplanted disease to the disease microenvironment can also be expressed in the above-mentioned cells to be transplanted (such as T cells) with reduced immunogenicity.

[0640] For another example, in order to increase the immunostimulatory activity of cells to be transplanted (such as T cells), immunostimulatory molecules can be expressed in the above-mentioned cells to be transplanted (such as T cells) with reduced immunogenicity.

[0641] On the other hand, the present application also provides a method for introducing NK elements, especially optimized recombinant NK elements, into the cells to be transplanted (such as UT cells) provided in the present application, so that T cells can recognize all virus-infected cells and tumor cells as efficiently and broadly as NK cells. Optionally, by further optimizing the transduction elements, CNK-T cells can be efficiently activated and kill tumor cells. Because NK targets include family member proteins such as MICA, MICB and ULBP1-6, which can be widely expressed in various types of tumor cells and cover different stages of tumor progression, CNK technology can effectively solve the off-target effects of single CAR-T and eliminate the chance of tumor immune escape. At the same time, the composite adapter (Chimeric Adaptor) introduced by CNK in the design can effectively transduce and amplify NK signals, transcend the limitations of immune checkpoints such as PD1 signals, efficiently activate T cells, and achieve the killing of tumor cells. The Chimeric Adapter amplifies CNK-T cell signals, counteracting immunosuppression in the tumor environment and activating T cells to kill tumor cells. Furthermore, CNK-T cells, through NK recognition of targets, can eliminate immunosuppressive cells such as MDSCs. After viral infection, cells express specific functional proteins that stimulate the assembly or transport of MHCI or directly promote the targeted degradation of MHCI molecules, thereby inhibiting the presentation of viral antigen epitopes and promoting immune escape.

[0642] On the one hand, the present disclosure provides a UT cell that also expresses a CNK receptor and, optionally, a CAR. Such UT cells may also be referred to as "CNKT-UT." In this application, it is verified that CNK-UT has a broad spectrum of tumor recognition and killing capabilities. This application further designs a composite specific target CAR / CNK-UT product, demonstrating that the CAR / CNK-UT product has a more powerful killing and activation function on tumor cells than conventional CAR-T; in animal experiments, the CAR / CNK-UT product also has a more efficient tumor clearance ability. UT technology realizes the transformation of allogeneic universality, and T cells are derived from healthy donors, thereby realizing the standardization and large-scale production of CNK-UT products, which can be prepared in advance and ensure the function of T cells to kill and activate tumor cells.

[0643] In some preferred embodiments, the CNKT-UT comprises a nucleic acid molecule encoding an amino acid sequence having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO. 158, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater identity. In some preferred embodiments, the CNKT-UT comprises a nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO. 158. In another aspect, the present disclosure also provides the use of CNKT-UT in the preparation of a medicament for treating a disease.

[0644] On the one hand, the present disclosure further provides a kind of chimeric protein construct described in the present application, encoding its nucleic acid molecule or immune cell of carrier, wherein the chimeric protein construct includes ERAD mechanism protein binding domain and targeting domain, and the targeting domain includes specific targeting or binding domain of target protein, and the target protein is expressed on the immune cell and on the target cell. In certain embodiments, the immune cell further includes or expresses the binding domain or molecule (such as CAR) targeting the target protein, encoding its nucleic acid molecule or carrier. In certain embodiments, the examples of such target proteins include CD123, CD5, CD7, CD38 or CD4.

[0645] Without being limited by theory, some target proteins are expressed both on target cells (such as cancer cells or virus-infected cells) and on immune cells for treatment. Such targets are usually difficult to treat by cell therapy methods, so when CAR targeting such targets is expressed on immune cells for treatment, CAR will also lead to the same target expressed by the immune cells themselves, causing autophagy in the immune cells, and then unable to amplify normally, or having limited effect on target cell killing. By the chimeric protein construct provided by the application, the target protein expressed by the immune cells themselves can be degraded, thereby avoiding autophagy, while retaining the specific killing effect on the target cells. Another advantage of using the chimeric protein construct of the application is that the chimeric protein construct targeting the target and the binding domain (such as CAR, TCR, etc.) targeting the target can be expressed simultaneously by co-expression, which is simpler and more effective than methods such as traditional gene knockout.

[0646] In certain embodiments, the chimeric protein construct further comprises a protein degradation pathway member (e.g., E3 ubiquitin ligase, proteasome, lysosome) binding domain as described herein. Any protein degradation pathway member binding domain described herein can be used.

[0647] Methods of administering cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, methods for adoptive T cell therapy are described in, for example, U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10):928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9; Davila et al. (2013) PLoS ONE 8(4):e61338.

[0648] In certain embodiments, cells or individual populations or subtypes of cells are administered to a subject in the range of about 1 million to about 100 billion cells, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, or a range defined by any two of the foregoing values), or about 100 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, or a range defined by any two of the foregoing values). In some embodiments, the number of cells present in the cell culture medium is about 10 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, about 65 billion cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases, about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value therebetween.

[0649] In some embodiments, the dose of total cells and / or the dose of individual subpopulations of cells is at or about 10 4 cells / kg body weight to at or around 10 9 cells / kg body weight, for example, in the range of 10 5 and 10 6 cells / kg body weight, e.g., at least or at least about or at or about 1×10 5 cells / kg, 1.5×10 5 cells / kg, 2×105 cells / kg or 1×10 6 cells / kg body weight. For example, in some embodiments, the cells are expressed at or about 10 4 and at or about 10 9 T cells / kg body weight or within a certain error range thereof, for example, between 10 5 and 10 6 T cells / kg body weight, such as at least or at least about or at or about 1×10 5 T cells / kg, 1.5×10 5 T cells / kg, 2×10 5 T cells / kg, or 1×10 6 T cells / kg body weight.

[0650] Cell can be used by any suitable mode, for example, by bolus, by injection, for example intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, through septal injection, subscleral injection, intrachoroidal injection, intracameral injection, injection under perineum, subconjunctival injection, under Tenon's fascia capsule (sub-Tenon) injection, retrobulbar injection, peribulbar injection, or posterior sclera (posterior juxtascleral) delivery. In some embodiments, they are used by parenteral, intrapulmonary and intranasal administration, and if local treatment is desired, then by intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In some embodiments, given dose is used by single bolus administration cell to use. In some embodiments, it is used by repeatedly bolus administration cell to use, for example, during being no more than 3 days, or by continuous infusion administration cell to use.

[0651] In some embodiments, a repeated dose method is provided, in which a first dose of cells is administered, followed by one or more second continuous doses. When administered to a subject using an adoptive therapy approach, the timing and size of multiple doses of cells are typically designed to increase the efficacy and / or activity and / or function of T cells expressing antigens (e.g., T cells expressing CAR). In some embodiments, repeated administration reduces the downregulation or inhibitory activity that may occur when inhibitory immune molecules such as PD-1 and / or PD-L1 are upregulated on T cells expressing antigens such as CAR. The method includes administering a first dose, typically followed by one or more continuous doses, and having a specific time frame between different doses.

[0652] In the context of adoptive cell therapy, the administration of a given "dose" includes the administration of a given amount or quantity of cells as a single composition and / or a single uninterrupted administration (e.g., as a single injection or continuous infusion), and also includes the administration of a given amount or quantity of cells provided with multiple individual compositions or infusions as a fractionated dose over a specified time period (no more than 3 days). Therefore, in some cases, the first or continuous dose is a single or continuous administration of a specified number of cells given or started at a single time point. However, in some cases, the first or continuous dose is administered with multiple injections or infusions over a period of no more than three days, such as three or two days, once a day, or multiple infusions over a single day.

[0653] Cell therapy involves genetically engineering cells (such as immune cells) to present tumor-associated antigens or express receptors that specifically recognize diseased cells. These cells are then exponentially expanded in vitro and then infused back into the patient's body, activating the immune system to attack tumor cells or directly and specifically recognize and kill them. In 2012, CD19-targeted CAR-T cells achieved the first-ever targeted elimination of tumor cells in a B-cell leukemia patient, becoming the next truly curative leukemia treatment after bone marrow stem cell transplantation and ushering in a new era of precision medicine cell therapy. This technology has the potential to be applied to the treatment of various hematologic malignancies and solid tumors. However, the current clinical efficacy of conventional CAR-T in treating solid tumors is poor. The reasons are: (1) The killing function of CAR-T is highly dependent on the recognition of tumor-associated antigens (TAA) by the CAR structure, but due to the heterogeneity of solid tumors, there are great differences in the expression of target proteins on the surface of tumor cells; currently, single-target CAR-T cannot completely recognize and kill all tumor cells, which leads to tumor immune escape and recurrence and metastasis; (2) The direct immunosuppressive effect of solid tumors, expressing PD-L1, B3H7, etc., directly inhibits the activation of T cells; (3) The immunosuppressive microenvironment of solid tumors contains a large number of immunosuppressive cells, such as TAM, Treg, and MDSC. It is difficult for conventional CAR-T to break through the limitations of immunosuppressive cells inside the tumor; (4) Conventional CAR-T is a personalized cell therapy. The T cells come from the patient himself. If the patient has received a lot of radiotherapy and chemotherapy, and the immune system function is damaged, it is difficult to isolate a sufficient number of T cells from the patient's peripheral blood. Even if they are isolated and transformed, the proliferation and killing function of T cells are still very weak, so it is difficult to achieve a therapeutic effect.

[0654] The cells to be transplanted (eg, T cells) provided in this application can overcome the problems faced in existing cell therapy, such as differences in tumor targets, tumor immunosuppressive environment, and sources of personalized therapeutic cells.

[0655] For the purpose of cell therapy, the cells to be transplanted (e.g., T cells, further, universal T cells with defects in TCRαβ and MHC molecule surface expression) with reduced immunogenicity can be further genetically engineered, for example, to express the co-expression portion as described above, to achieve therapeutic purposes.

[0656] For example, in order to enable the cells to be transplanted (such as T cells) to specifically act on disease-related target proteins or cells expressing disease-related target proteins, binding domains or molecules that recognize or bind to disease-related target proteins can be expressed in the above-mentioned cells to be transplanted (such as T cells) with reduced immunogenicity, such as chimeric antigen receptors CAR, engineered TCR, or CNK receptors provided in this application, or antiviral protein binding domains, etc.

[0657] In some embodiments, molecules (such as chemokine receptors or cytokine receptors) that can ...

Claims

1. A fusion protein comprising a module that binds to an antigen and a module that promotes trimer formation, wherein the antigen comprises a cell membrane receptor protein that can achieve clathrin-dependent endocytosis (CDE). 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 such as monocytes; Preferably, the T cell membrane receptor antigen is selected from one or more of TCR, CD3, CD4, CD5, CD7, CCR5, and CXCR4; Preferably, the B cell membrane receptor antigen is selected from CD20 and CD22; Preferably, the membrane receptor antigen of the antigen-presenting cells such as monocytes is selected from Fc receptor antigens; preferably, the Fc receptor antigen is selected from one or more of Fcα, FcγRI, FcγRII, FcγRIV, FcεRI and Fcα / μR; preferably, Fcα is FcαRI, more preferably CD89; preferably, FcγRI is CD64; preferably, FcγRII is FcγRIIB2 (CD32); preferably, FcεRI is CD89.

2. The fusion protein according to claim 1, wherein the component promoting trimer formation is selected from adiponectin or its collagen-like domain, T4 fibrin trimer domain, trimeric structure of human collagen, such as: the amino-terminal NC1 domain of transmembrane collagens XIII, XV, XVII and XVIII, the NC2 domain of fibril-associated collagens IX, XII, XIV, XVI, XIX, XX and XXI, the GCN4 trimerization domain, human lung 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 the complementary heptad repeat region of enveloped virus class I fusion protein; Preferably, the component promoting trimer formation comprises the amino-terminal NC1 domain of XV; Preferably, the component that promotes trimer formation comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 18, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; preferably, its amino acid sequence is the amino acid sequence shown in SEQ ID NO. 18 or an amino acid sequence obtained by deleting, adding or substituting 1, 2 or 3 residues in SEQ ID NO:

18.

3. The fusion protein according to claim 1 or 2, wherein the component that binds to the antigen is selected from an antibody or an antigen-binding fragment thereof to the antigen or a ligand that binds to the membrane receptor protein.

4. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof for the TCR antigen comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO.1 or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence SEQ ID NO.2 or any variant thereof, and a heavy chain CDR3 selected from the amino acid sequence SEQ ID NO.3 or any variant thereof; and / or a light chain CDR1 selected from the amino acid sequence SEQ ID NO.4 or any variant thereof, a light chain CDR2 selected from the amino acid sequence SEQ ID NO.5 or any variant thereof, and a light chain CDR3 selected from the amino acid sequence SEQ ID NO.6 or any variant thereof.

5. The fusion protein according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof for the TCR antigen comprises heavy chain CDR1, CDR2, and CDR3 with amino acid sequences as shown in SEQ ID NOs: 1, 2, and 3, and / or comprises light chain CDR1, CDR2, and CDR3 with amino acid sequences as shown in SEQ ID NOs: 4, 5, and 6, respectively.

6. The fusion protein according to any one of claims 1 to 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 a light chain variable region selected from the amino acid sequence SEQ ID NO. 8 or any variant thereof.

7. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof to the CD3 antigen comprises a heavy chain CDR1 selected from the amino acid sequence of SEQ ID NO. 9 or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence of SEQ ID NO. 10 or any variant thereof, and a heavy chain CDR3 selected from the amino acid sequence of SEQ ID NO. 11 or any variant thereof; and / or a light chain CDR1 selected from the amino acid sequence of SEQ ID NO. 12 or any variant thereof, a light chain CDR2 selected from the amino acid sequence of SEQ ID NO. 13 or any variant thereof, and a light chain CDR3 selected from the amino acid sequence of SEQ ID NO. 14 or any variant thereof.

8. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof to the CD3 antigen comprises heavy chain CDR1, CDR2, and CDR3 with amino acid sequences as shown in SEQ ID NOs: 9, 10, and 11, and / or light chain CDR1, CDR2, and CDR3 with amino acid sequences as shown in SEQ ID NOs: 12, 13, and 14, respectively.

9. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof against the CD3 antigen comprises a heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 15 or any variant thereof, and / or a light chain variable region selected from the amino acid sequence of SEQ ID NO. 16 or any variant thereof.

10. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof to the CD5 antigen comprises a heavy chain CDR1 selected from the amino acid sequence of SEQ ID NO.215 or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence of SEQ ID NO.216 or any variant thereof, and a heavy chain CDR3 selected from the amino acid sequence of SEQ ID NO.217 or any variant thereof; and / or a light chain CDR1 selected from the amino acid sequence of SEQ ID NO.218 or any variant thereof, a light chain CDR2 selected from the amino acid sequence of SEQ ID NO.219 or any variant thereof, and a light chain CDR3 selected from the amino acid sequence of SEQ ID NO.220 or any variant thereof.

11. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof to the CD5 antigen comprises heavy chain CDR1, CDR2, and CDR3 with amino acid sequences as shown in SEQ ID NOs: 215, 216, and 217, and / or comprises light chain CDR1, CDR2, and CDR3 with amino acid sequences as shown in SEQ ID NOs: 218, 219, and 220, respectively.

12. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof against the CD5 antigen comprises a heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 221 or any variant thereof, and / or a light chain variable region selected from the amino acid sequence of SEQ ID NO. 222 or any variant thereof.

13. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof to the CD7 antigen comprises a heavy chain CDR1 selected from the amino acid sequence of SEQ ID NO. 223 or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence of SEQ ID NO. 224 or any variant thereof, and a heavy chain CDR3 selected from the amino acid sequence of SEQ ID NO. 225 or any variant thereof; and / or a light chain CDR1 selected from the amino acid sequence of SEQ ID NO. 226 or any variant thereof, a light chain CDR2 selected from the amino acid sequence of SEQ ID NO. 227 or any variant thereof, and a light chain CDR3 selected from the amino acid sequence of SEQ ID NO. 228 or any variant thereof.

14. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof to the CD7 antigen comprises heavy chain CDR1, CDR2, and CDR3 with amino acid sequences as shown in SEQ ID NOs: 223, 224, and 225, and / or comprises light chain CDR1, CDR2, and CDR3 with amino acid sequences as shown in SEQ ID NOs: 226, 227, and 228, respectively.

15. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof against the CD7 antigen comprises a heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 229 or any variant thereof, and / or a light chain variable region selected from the amino acid sequence of SEQ ID NO. 230 or any variant thereof.

16. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof to the CD4 antigen comprises a heavy chain CDR1 selected from the amino acid sequence of SEQ ID NO. 231 or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence of SEQ ID NO. 232 or any variant thereof, and a heavy chain CDR3 selected from the amino acid sequence of SEQ ID NO. 233 or any variant thereof; and / or a light chain CDR1 selected from the amino acid sequence of SEQ ID NO. 234 or any variant thereof, a light chain CDR2 selected from the amino acid sequence of SEQ ID NO. 235 or any variant thereof, and a light chain CDR3 selected from the amino acid sequence of SEQ ID NO. 235 or any variant thereof.

17. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof to the CD4 antigen comprises heavy chain CDR1, CDR2, and CDR3 with amino acid sequences as shown in SEQ ID NOs: 231, 232, and 233, respectively, and / or comprises light chain CDR1, CDR2, and CDR3 with amino acid sequences as shown in SEQ ID NOs: 234, 235, and 236, respectively.

18. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof against the CD4 antigen comprises a heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 237 or any variant thereof, and / or a light chain variable region selected from the amino acid sequence of SEQ ID NO. 238 or any variant thereof.

19. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof to the CD20 antigen comprises a heavy chain CDR1 selected from the amino acid sequence of SEQ ID NO. 239 or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence of SEQ ID NO. 240 or any variant thereof, and a heavy chain CDR3 selected from the amino acid sequence of SEQ ID NO. 241 or any variant thereof; and / or a light chain CDR1 selected from the amino acid sequence of SEQ ID NO. 242 or any variant thereof, a light chain CDR2 selected from the amino acid sequence of SEQ ID NO. 243 or any variant thereof, and a light chain CDR3 selected from the amino acid sequence of SEQ ID NO. 244 or any variant thereof.

20. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof to the CD20 antigen comprises the heavy chain CDR1, CDR2, and CDR3 of the amino acid sequences shown in SEQ ID NOs: 239, 240, and 241, respectively, and / or comprises the light chain CDR1, CDR2, and CDR3 of the amino acid sequences shown in SEQ ID NOs: 242, 243, and 244, respectively.

21. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof against the CD20 antigen comprises a heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 245 or any variant thereof, and / or a light chain variable region selected from the amino acid sequence of SEQ ID NO. 246 or any variant thereof.

22. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof to the CD22 antigen comprises a heavy chain CDR1 selected from the amino acid sequence of SEQ ID NO. 247 or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence of SEQ ID NO. 248 or any variant thereof, and a heavy chain CDR3 selected from the amino acid sequence of SEQ ID NO. 249 or any variant thereof; and / or a light chain CDR1 selected from the amino acid sequence of SEQ ID NO. 250 or any variant thereof, a light chain CDR2 selected from the amino acid sequence of SEQ ID NO. 251 or any variant thereof, and a light chain CDR3 selected from the amino acid sequence of SEQ ID NO. 252 or any variant thereof.

23. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof to the CD22 antigen comprises the heavy chain CDR1, CDR2, and CDR3 of the amino acid sequences shown in SEQ ID NOs: 247, 248, and 249, respectively, and / or comprises the light chain CDR1, CDR2, and CDR3 of the amino acid sequences shown in SEQ ID NOs: 250, 251, and 252, respectively.

24. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof against the CD22 antigen comprises a heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 253 or any variant thereof, and / or a light chain variable region selected from the amino acid sequence of SEQ ID NO. 254 or any variant thereof.

25. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof to the CD64 antigen comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO.255 ​​or any variant thereof, a heavy chain CDR2 selected from the amino acid sequence SEQ ID NO.256 or any variant thereof, and a heavy chain CDR3 selected from the amino acid sequence SEQ ID NO.257 or any variant thereof; and / or a 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, and a light chain CDR3 selected from the amino acid sequence SEQ ID NO.260 or any variant thereof.

26. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof against the CD64 antigen comprises heavy chain CDR1, CDR2, and CDR3 with amino acid sequences as shown in SEQ ID NOs: 255, 256, and 257, respectively, and / or comprises light chain CDR1, CDR2, and CDR3 with amino acid sequences as shown in SEQ ID NOs: 258, 259, and 260, respectively.

27. The fusion protein according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof against the CD64 antigen comprises a heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 261 or any variant thereof, and / or a light chain variable region selected from the amino acid sequence of SEQ ID NO. 262 or any variant thereof.

28. The fusion protein according to any one of claims 1 to 27, wherein the antigen-binding component and the trimer-forming promoting component can be directly connected or connected via a linker L1; Preferably, the joint is a flexible joint; Preferably, the fusion protein further comprises a purification tag; the purification tag is at the N-terminus, C-terminus, or between the antigen-binding component and the component that promotes trimer formation of the fusion protein; more preferably, the purification tag is between the antigen-binding component and the component that promotes trimer formation; Preferably, the purification tag is selected from the group consisting of a GST tag, a His tag, a Myc tag, an E tag, a Strep tag, and an HA tag; more preferably, the Strep tag is an STII tag, the amino acid sequence of which is shown in SEQ ID NO.17; Preferably, the flexible linker L1 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. 24). 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); 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. The fusion protein according to any one of claims 1 to 6, comprising an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 36, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or 99% or more identical; preferably, the amino acid sequence is the amino acid sequence shown in SEQ ID NO: 36 or an amino acid sequence obtained by deleting, adding or substituting 1, 2 or 3 residues in SEQ ID NO:

36.

30. A nucleic acid molecule encoding the fusion protein of any one of claims 1-29.

31. A vector comprising the nucleic acid molecule of claim 30.

32. A cell comprising the nucleic acid molecule of claim 30 or the vector of claim 31.

33. 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. A prodrug comprising any one selected from the following: (1) The fusion protein and LNP of any one of claims 1 to 26; Preferably, the fusion protein and the LNP are connected via a linker L2; more preferably, the linker L2 is a polypeptide having a cysteine ​​residue added to the C-terminus of a flexible polypeptide; 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), GGGGSGGGGSGGGGS (SEQ ID NO.25), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO.26), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO.27), GSGSGSGS (SEQ ID NO.28), GGSGSGSG (SEQ ID NO.29), GGSGSG (SEQ ID NO.30), GGSGSG (SEQ ID NO.31), GGSGSG (SEQ ID NO.32), GGSGSG (SEQ ID NO.33), GGSGSG (SEQ ID NO.34), GGSGSG (SEQ ID NO.35), GGGGSGGGSGGGGSGGGGS (SEQ ID NO.36), GGGGSGGGGSGGGGS (SEQ ID NO.37), GGSGSGS (SEQ ID NO.38), GGSGSGS (SEQ ID NO.39), GGSGS NO.30), GGSG (SEQ ID NO.31); 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); 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 small molecule compound is a chemotherapeutic drug; More preferably, the biologically active nucleic acid is selected from one or more 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 a nucleic acid encoding a chimeric protein construct; More preferably, the biologically active protein is selected from one or more of a chimeric antigen receptor (CAR), a chimeric natural killer cell receptor (CNK) complex, a pro-apoptotic protein, and a chimeric protein construct; More preferably, the small molecule compound is selected from one or more chemotherapeutic drugs; Preferably, wherein The pro-apoptotic proteins are Bcl-2 family proteins, including but not limited to Bax, Bak, Bok, Bad, Bid, Bik, Bim, HrkBnip3, Nix / Bnip3L, Noxa and Puma; Preferably, the nucleic acid having biological activity 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 receptor (CAR), chimeric natural killer cell receptor (CNK) complex or chimeric protein construct; Preferably, the biologically active nucleic acid is an expression vector; Preferably, the chemotherapy drug contained in the LNP is selected from one or more of: cytotoxins, alkylating agents, podophyllums, camptothecins, taxanes, antimetabolites, antibiotic anti-tumor drugs, examples of which include but are not limited to imidazole tetrazinones, such as temozolomide; platinum drugs, such as oxaliplatin, cisplatin, carboplatin, nedaplatin, dicycloplatin, levoplatin, triplatin tetranitrate, phenanthreneplatin, picoplatin, satraplatin; nitrosoureas, such as carmustine, cyclohexyl nitrosourea, methyl nitrosourea, pyrimidine nitrosourea, carmustine , lomustine, fotemustine, nimustine, ranimustine, streptozotocin; camptothecins, such as camptothecin, hydroxycamptothecin, irinotecan, topotecan; vinca alkaloids, such as vinorelbine, vinblastine, vincristine, vindesine, vinblastine; procarbazine; mitoxantrone hydrochloride; nitrogen mustards, such as mechlorethamine, chlorambucil, cyclophosphamide, ifosfamide, estradiol mustard, trofosfamide, phenylalanine mustard, chlorambucil, melphalan, prednimustine, bendamustine, uracil, estramustine, thiothiazolinone, Tipa; alkyl sulfonates such as busulfan, mannosulfur, and suosulfur; fluoropyrimidine derivatives such as gemcitabine, capecitabine, fluorouracil, fufuroxil, doxifluridine, tegafur, carmofur, and trifluridine; taxanes such as paclitaxel, albumin-bound paclitaxel, liposomal paclitaxel, and docetaxel; one or two or three of pemetrexed, etoposide, irinotecan, mitomycin, cytarabine, azacitidine, amrubicin, methotrexate, epirubicin, doxorubicin, sapacitabine, plinabulin, triazolam, dipivefrin hydrochloride, tegafur, and encequidar; Preferably, the cytotoxin contained in the LNP is selected from dolastatin and auristatin cytotoxic molecules, and maytansine cytotoxic molecules; the DNA damaging agent includes but is not limited to calicheamicin, duocarmycin, anthramycin derivative PBD, and camptothecin derivative SN-38; More preferably, the cytotoxin contained in the LNP is selected from amanitins, anthracyclines, baccatins, camptothecins, cemadotins, colchicines, colcimids, combretastatins, cryptophycins, discodermolides, docetaxel, doxorubicin, ), echinomycins, eleutherobins, epothilones, estramustines, lexitropsins, maytansines, methotrexate, netropsins, puromycins, rhizoxins, taxanes, tubulysins, or vinca alkaloids; (2) The fusion protein of any one of claims 1 to 26 and a cytotoxin, wherein the fusion protein and the cytotoxin are connected via a linker unit; Preferably, the cytotoxin connected to the fusion protein via a linker unit is selected from dolastatin, auristatin, and maytansine cytotoxic molecules; the DNA damaging agent includes but is not limited to calicheamicin, duocarmycin, anthramycin derivative PBD, and camptothecin derivative SN-38; Preferably, the cytotoxin connected to the fusion protein via a linker unit is selected from the group consisting of amanitins, anthracyclines, baccatins, camptothecins, cemadotins, colchicines, colcimids, combretastatins, cryptophycins, discodermolides, docetaxel, doxorubicin, cin), echinomycins, eleutherobins, epothilones, estramustines, lexitropsins, maytansines, methotrexate, netropsins, puromycins, rhizoxins, taxanes, tubulysins, or vinca alkaloids; More preferably, the linker unit is selected from the group consisting of 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);, (3) The fusion protein and a marker according to any one of claims 1 to 26; Preferably, the label is selected from iron oxide nanoparticles, UV-visible labels, near-infrared labels, luminescent groups, phosphorescent groups, magnetic spin resonance labels, photosensitizers, photocleavable parts, chelating centers, heavy atoms, radioisotopes, isotope detectable spin resonance labels, paramagnetic parts, chromophores or combinations thereof; more preferably, the label is iron oxide nanoparticles.

35. The prodrug according to claim 34, wherein the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, a costimulatory signaling region and an intracellular signaling domain, wherein the antigen bound by the extracellular domain is selected from the group consisting of mesothelin (MSLN), B7-H3 (CD276), chondroitin sulfate proteoglycan 4 (CSPG4), Muc 16, Claudin 18.2, Claudin 8, NY-ESO-1, CD19, CD22, CD23, myeloproliferative leukemia protein 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); 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 molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, 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.

36. The prodrug of claim 34, wherein the chimeric natural killer cell receptor (CNK) complex comprises the following components: (1) An NK cell activating receptor component, comprising at least an NK cell activating receptor or a functional variant thereof, wherein the NK cell activating receptor comprises: (a) an NK cell activating receptor extracellular domain (ED) or a functional variant thereof, (b) an NK cell activating receptor transmembrane domain (TMD) or a functional variant thereof, and (c) an NK cell activating receptor intracellular domain (ICD) or a functional variant thereof; optionally, a hinge or linker is contained between the NK cell activating receptor extracellular domain or a functional variant thereof, the NK cell activating receptor transmembrane domain or a functional variant thereof, and / or the NK cell activating receptor intracellular domain or a functional variant thereof; (2) A CNK signal transduction component, comprising at least (i) an NK cell signal transducer (adaptor) or a functional variant thereof, wherein 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, a hinge or linker is contained between the NK cell signal transducer extracellular domain or a functional variant thereof, 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; and Optionally, a hinge or linker is included between the NK activating receptor component and the CNK signal transduction component.

37. The prodrug according to claim 34, wherein the chimeric protein construct comprises an endoplasmic reticulum-associated degradation (ERAD) machinery protein binding domain and a targeting domain; More preferably, 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; 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, other viral endoplasmic reticulum resident proteins of Cowpox Virus protein CPXV203; More preferably, the aforementioned chimeric protein construct further comprises a protein degradation pathway member (e.g., E3 ubiquitin ligase, proteasome, lysosome) binding domain, and optionally, the protein degradation pathway member binding domain is connected to the ERAD mechanism protein binding domain; More preferably, the targeting domain comprises an antibody or a functional fragment thereof (e.g., Fd, Fv, Fab, Fab', F(ab')2, Fv (scFv), single-chain antibody (scFv), nanobody, diabody, triabody and tetrabody) that specifically targets a target protein; The target protein is a pathogenic protein. Optionally, the pathogenic protein is a tumor-related protein, a virus-related protein, an immune function-related protein (including an immunosuppressive protein and an immune-activating protein), an autoantigen protein, or a neurodegenerative disease-related protein; The autoantigen protein is selected from the group consisting of autoantigens associated with type I diabetes, such as islet cell antigen (ICA), insulin (IAA), glutamic acid decarboxylase 65 (GAD65), and insulinoma antigen-2 (IA-2); autoantigens associated with rheumatoid arthritis (RA), such as citrullinated protein / peptide antibodies, heterogeneous nuclear ribonucleoprotein A2 / B1, aldolase, alpha-enolase, calreticulin, heat-activated protein (HSP60), BiP, PGK1, stress-induced phosphoprotein 1, and FUSE-BP1 / 2; autoantigens associated with systemic lupus erythematosus (SLE), such as deoxyribonucleoprotein, SmD1 and SmD3, Clq, sore anticoagulant (LA), cardiolipin (CL), β2 glycoprotein I (β2 GP I), prothrombin (PT) and phosphatidylserine (PS); autoantigens associated with systemic sclerosis (SSc) / scleroderma (SD), such as Scl-70, SSA, Ro52; antigens associated with autoimmune liver disease, such as mitochondrial antigens, Sp100, PML, gp210, p62; autoantigens associated with myasthenia gravis, such as acetylcholine receptor; autoantigens associated with central nervous system autoimmune diseases (limbic encephalitis, encephalomyelitis, cerebellar ataxia), such as voltage-gated potassium channel (VGKC) complex, voltage-gated calcium channel receptor, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor, γ-aminobutyric acid-B (GABAB) receptor, glycine receptor; autoantigens associated with multiple sclerosis (MS) Autoantigens related to polymyositis (PM) and dermatomyositis (DM) include Jo-1, Mi-2, PM-Scl, and Ro-52; autoantigens related to gluten-sensitive enteropathy include endomysial antibodies (EMA) and tissue transglutaminase (tTG); autoantigens related to anti-NMDAR antibody encephalitis include N-methyl-D-aspartate receptor; autoantigens related to neuromyelitis optica (NMO) include aquaporin 4 (AQP4); autoantigens related to reproduction include ovarian antigens and sperm antigens; 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, BR CA1, 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, T ANI, 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, TGF BR2, 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, D CC, DPC4 / SMAD4, E-CAD, E2F1 / RBAP, ELK1, ELK3, EPH, EPHA1, E2F1, EPHA3, ERG, ETS1, ETS2, LMO-1, LMO-2, L-MYC, LYL1, LYT-10, MDM-2, MLH1, MLL, MLM, N-MYC, OST, PAX-5, PMS-1, FGF4, FGF6, FANCA, FLI1 / ERGB2, FOSL1, FOSL2, GLI, HRAS1, HRX / MLLT1, HRX / MLLT2, KRAS2, MADH4, MASI, MCF2, MLLT1 / MLL, MLLT2 / HRX, MTG8 / RUNX1, MYCLK1, MYH11 / CBFB, NFKB2, NOTCH1, NPM1 / ALK, NRG / REL, NTRK1, PBX1 / TCF3, PML / RARA, PRCA1, RUNX1, RUNX1 / CBFA2T1, SET, SHP2, TCF3 / PBX1, TNFα, Clusterin, Survivin, TOEβ, c-fos, c-SRC, estrogen receptor ER-α, androgen receptor ER-α. receptor, AR), and INT-1; optionally, the tumor-associated protein is selected from the following group: Bcl-2 family members (such as Bcl-2, Bcl-xL and Bcl-w), VEGF / VEGFR, PDGFRβ, EGFR, EGFR mutants, IGF-1R, HDACs, HER2, MYC, KRAS, AFP, CEA, CA199, estrogen receptor (estrogen receptor ER-α), androgen receptor (Androgen receptor, AR), tyrosine kinases (c-ABL, BCR-ABL, BTK, FAK, PTK6, Wee1, TRK transmembrane receptors), serine / threonine kinase receptors (IRAK4, LRRK2, B-Raf, RIPK2, CDK4 / 6, CDK7, CDK8, CDK8 / 19, CDK9, TBK1), protein kinase II (CK2), epigenetic-related proteins (BRD2, BRD3, BRD4, BRDT, TRIM24, BRD9, PBRM1, SMARCA2, SMARCA4, EP300, EZH2, WDR5), adrenomedullin (ADM), DPP3; The virus-associated protein is selected from the group consisting of: HBV surface antigen, HBV capsid glycoprotein, HBeAg, HBV DNA polymerase, HBV encoded X protein (HBx), HIV Gag protein, HIV Env protein, HIV gp120, HIV-1 reverse transcriptase, HIV gp120, HCV NS3-4A protease, HCV RNA polymerase, HCV envelope protein, EBV DNA polymerase, EBV EBNA1, coronavirus RNA synthetase, coronavirus spike protein, coronavirus envelope protein, coronavirus membrane protein, coronavirus nucleocapsid protein, RNA-dependent RNA polymerase (RdRp), such as SARS-CoV-2 RNA-dependent RNA polymerase, herpesvirus DNA and RNA polymerase, herpesvirus capsid glycoprotein, CMV DNA polymerase, CMV capsid glycoprotein, RSV surface protein, RSV capsid protein, RSV RNA polymerase, influenza virus RNA polymerase, influenza virus envelope protein, HPV DNA polymerase, and HPV capsid protein; The immune function-related protein is selected from the group consisting of: 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.), and immune stimulatory / co-stimulatory molecules (such as CD3, CD80 / 86, CD28, etc.); The target protein associated with a nervous system disease is selected from the group consisting of: Tau, amyloid-β (Aβ), α-synuclein, mutant huntingtin (mHTT), α-synuclein, TAR RNA binding protein (TARDBP) and FUS RNA binding protein (FUS); The aforementioned chimeric protein construct is linked to at least one co-expression portion, the co-expression portion being independently selected from: a complete viral ER-resident glycoprotein (e.g., HCMV US2, US3, US11, US10, adenovirus E3-K1 9, HCMV US6, HSV ICP47), a chimeric antigen receptor (CAR), a functional T cell receptor (TCR), a chemokine receptor, and a NK cell activating receptor; The chemokine receptor is selected from the group consisting of: CCR4, CCR5, CCR6, CCR7, CCR9, CCR2b, CXCR1, CXCR2 and CXCR4; The NK cell activating receptor comprises: (a) an NK cell activating receptor extracellular domain (ED) or a functional variant thereof, (b) an NK cell activating receptor transmembrane domain (TMD) or a functional variant thereof, and (c) an NK cell activating receptor intracellular domain (ICD) or a functional variant thereof; optionally, a hinge or linker is contained between the NK cell activating receptor extracellular domain or a functional variant thereof, the NK cell activating receptor transmembrane domain or a functional variant thereof, and / or the NK cell activating receptor intracellular domain or a functional variant thereof; 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. Use of 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 the prodrug according to any one of claims 34 to 37 in the preparation of a drug or kit for diagnosing, treating or preventing cancer or viral infections; 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, liver metastasis of colon cancer, 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, B-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 cancer, gastric cancer, pancreatic cancer, triple-negative invasive breast cancer, renal cell carcinoma, squamous cell lung cancer cancer, hepatocellular carcinoma, urothelial carcinoma, leukemia, B-cell leukemia, B-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, adult acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, childhood acute lymphoblastic leukemia, refractory childhood acute lymphoblastic leukemia, acute leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, prolymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, relapsed plasma cell myeloma, refractory plasma cell myeloma, multiple myeloma, relapsed or refractory multiple myeloma, multiple myeloma of bone, malignant glioma of brain, myelodysplastic syndrome, EGFR-positive colorectal cancer, glioblastoma multiforme, neoplasms, blastic plasmacytoid dendritic cell neoplasm, liver metastasis, solid tumors, advanced solid tumors, mesothelin-positive tumors, hematological malignancies and other advanced malignancies; Preferably, the viral infection diseases include respiratory viral diseases, gastrointestinal viral diseases, liver viral diseases, skin and mucous membrane viral diseases, eye viral diseases, central nervous system viral diseases, lymphocytic viral diseases, insect-borne viral diseases, slow viral infection diseases, disease, etc. More preferably, respiratory viral diseases include infections caused by rhinovirus, adenovirus, respiratory syncytial virus, parainfluenza virus, and coronavirus; influenza; mumps; More preferably, the gastrointestinal viral diseases include poliomyelitis; Kuksaki virus infection; ECHO virus infection; viral gastroenteritis including rotavirus gastroenteritis, Norwalk virus gastroenteritis, adenovirus gastroenteritis, astrovirus gastroenteritis, coronavirus gastroenteritis and calicivirus gastroenteritis; More preferably, the viral liver diseases include hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, Epstein-Barr virus hepatitis and cytomegalovirus hepatitis; More preferably, viral diseases of the skin and mucous membranes include measles, rubella, roseola infantum, chickenpox and herpes zoster, smallpox, herpes simplex virus infection, rabies and foot-and-mouth disease; More preferably, the ocular viral diseases include epidemic keratoconjunctivitis, follicular conjunctivitis and herpes keratoconjunctivitis; 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 encephalitis and lymphocytic choriomeningitis; More preferably, the lymphocytic viral diseases include infectious mononucleosis, cytomegalovirus infection and acquired immunodeficiency syndrome; 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, Omsk hemorrhagic fever, Marburg disease and Ebola hemorrhagic fever; dengue fever and dengue hemorrhagic fever; West Nile fever; Colorado tick fever; sandfly fever; More preferably, the lentiviral infectious diseases include subacute sclerosing panencephalitis, kuru, progressive multifocal leukoencephalopathy and subacute spongiform encephalopathy (corticostriatal degeneration).

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