WO2024159695A1 - SynNotch受体及其用途 - Google Patents

SynNotch受体及其用途 Download PDF

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Publication number
WO2024159695A1
WO2024159695A1 PCT/CN2023/103857 CN2023103857W WO2024159695A1 WO 2024159695 A1 WO2024159695 A1 WO 2024159695A1 CN 2023103857 W CN2023103857 W CN 2023103857W WO 2024159695 A1 WO2024159695 A1 WO 2024159695A1
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Prior art keywords
cell
receptor
cells
chimeric polypeptide
nucleic acid
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PCT/CN2023/103857
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English (en)
French (fr)
Inventor
王烃
张彩
胡渊
陈敏华
谢思奇
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Shanghai NK Cell Tech Co Ltd
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Shanghai NK Cell Tech Co Ltd
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Priority to EP23919260.2A priority Critical patent/EP4545557A4/en
Priority to JP2025504478A priority patent/JP7853514B2/ja
Priority to KR1020257002120A priority patent/KR20250028389A/ko
Publication of WO2024159695A1 publication Critical patent/WO2024159695A1/zh
Priority to US19/026,698 priority patent/US20250152631A1/en
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Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention relates to a SynNotch receptor and a use thereof. More specifically, the present invention relates to a use of an isolated polypeptide in preparing a SynNotch synthetic receptor, a chimeric polypeptide, a first nucleic acid molecule, a first expression vector, a recombinant cell, a pharmaceutical composition and a use thereof, a method for activating immune cells, and a method for tracking first cell-second cell contact.
  • the receptor system can achieve multiple combinations of different input signals and output signals, and multiple groups of systems in the same system (cell) are orthogonal; at the same time, it can operate in many cells and is universal; at the same time, it has many excellent characteristics such as simplicity and controllability, and is a powerful tool for using synthetic biology to transform cells.
  • the traditional Notch signaling pathway includes Notch's corresponding ligand, Notch molecules, and Notch-regulated downstream genes.
  • the Notch molecule is a transmembrane molecule, which can be divided into three parts: the extracellular structure, the transmembrane domain, and the intracellular domain.
  • the cell where the ligand is located undergoes endocytosis, pulling the Notch molecule, exposing the S2 protease cleavage site on the extracellular domain of the Notch molecule, which is cut by members of the metalloprotease ADAMs family; further, along with the cutting and cleavage of the extracellular domain, the intracellular domain of the Notch molecule is cut by ⁇ -secretase and released into the cell, and then enters the nucleus as a transcription factor to regulate the expression of downstream related genes.
  • the extracellular domain uses a single antibody chain that specifically recognizes the antigen, while the intracellular domain regulates the expression of pre-set target genes or factors.
  • Gene regulatory loops activated by different antigens can be designed in the same cell, and have good orthogonality.
  • the SynNotch system can be applied to many cell types, including neural cells, tumor cells, epidermal cells, and immune cells.
  • the combination of CAR technology and the SynNotch system in immune cell modification can achieve the "AND gate” activation of immune cells, that is, immune cells can only be activated when the cells express two specific surface antigens at the same time.
  • immune cells can secrete single-chain antibodies, cytokines, and other therapeutic factors after contacting specific antigens, and have anti-tumor effects both in vitro and in vivo. It can be seen that the SynNotch receptor system has great technical advantages in cell modification.
  • the present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent.
  • the present invention provides a SynNotch receptor, which can enhance its ability to activate downstream genes and reduce the background leakage activation level.
  • the SynNotch system is very powerful, but the currently commonly used initial version of the SynNotch system (also known as SynNotch synthetic receptor, SynNotch) has the following disadvantages: First, after the initial version of SynNotch is activated, its downstream transcriptional expression regulation ability is not very strong, that is, the expression level of activated downstream genes is not very high, which will limit the use of SynNotch, especially for situations where downstream genes need to have a high expression level to be effective. Second, the initial version of SynNotch has a high background leakage level. For genes that require strict regulation downstream, its high level of leakage expression may cause the SynNotch molecular switch to fail, and the molecular expression circuit will directly bypass the SynNotch molecular switch to achieve expression.
  • the inventor unexpectedly discovered through experiments that the key point of the above problem lies in the insufficiency of the transmembrane domain of the SynNotch molecule.
  • the initial version of the SynNotch synthetic receptor selected the transmembrane domain of the mouse-derived Notch molecule as the transmembrane domain of the SynNotch synthetic receptor.
  • the activation efficiency and leakage efficiency of the SynNotch synthetic receptors constructed using different transmembrane domains are bound to be different in the cells of different species.
  • the present invention provides a use of an isolated polypeptide in the preparation of a SynNotch synthetic receptor, wherein the isolated polypeptide has at least 80% identity with the transmembrane region of the Notch receptor protein from Xenopus tropicalis.
  • the inventors have found that the SynNotch synthetic receptor prepared by selecting the transmembrane region of the Notch receptor protein from Xenopus tropicalis can improve its ability and efficiency in activating transcription of downstream genes and reduce the background leakage of the SynNotch synthetic receptor.
  • a chimeric polypeptide in another aspect of the present invention, includes: an extracellular region, the extracellular region has the activity of binding to a first molecule; a transmembrane region, the N-terminus of the transmembrane region is connected to the C-terminus of the extracellular region; an intracellular region, the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region; wherein the transmembrane region includes an isolated polypeptide, and the isolated polypeptide has at least 80% identity with the transmembrane region of the Notch receptor protein derived from tropical clawed frog.
  • the chimeric polypeptide of the present invention selects the transmembrane region of the Notch receptor protein of tropical clawed frog as the transmembrane region of the chimeric polypeptide (i.e., the SynNotch synthetic receptor), which can improve the ability and efficiency of the chimeric polypeptide to activate downstream gene transcription, and reduce the background leakage of the chimeric polypeptide.
  • the present invention provides a first nucleic acid molecule.
  • the first nucleic acid molecule encodes the aforementioned chimeric polypeptide of claim 1.
  • the first nucleic acid molecule encodes the aforementioned chimeric polypeptide.
  • the present invention provides a first expression vector.
  • the first expression vector carries the aforementioned first nucleic acid molecule. After the first expression vector of the present invention is introduced into a suitable receiving cell, the aforementioned chimeric polypeptide can be expressed.
  • the present invention provides a recombinant cell.
  • the recombinant cell comprises: carrying the aforementioned first nucleic acid molecule or the aforementioned first expression vector; or, expressing the aforementioned chimeric polypeptide.
  • the recombinant cell of the present invention can effectively express the aforementioned chimeric polypeptide.
  • the present invention proposes a pharmaceutical composition.
  • the pharmaceutical composition includes the aforementioned chimeric polypeptide, the aforementioned first nucleic acid molecule, the aforementioned first expression vector or the aforementioned recombinant cell.
  • the cell expressing the above chimeric polypeptide can recognize the first molecule, which can achieve a variety of different signal output types after binding to the first molecule, such as activation and inhibition of expression of a specific gene, or the immune cell expressing the chimeric polypeptide can secrete factors with therapeutic effects (such as single-chain antibodies, cytokines, etc.) after contacting the first molecule, which is used for anti-tumor; and the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in cells (such as immune cells), and the chimeric polypeptide can be expressed on the aforementioned recombinant cells.
  • the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in cells (such as immune cells), and the chimeric polypeptide can be expressed on the aforementioned recombinant cells.
  • a pharmaceutical composition containing the aforementioned chimeric polypeptide, the first nucleic acid molecule, the first expression vector or the recombinant cell can target the first molecule for the prevention and/or treatment of related diseases, such as for the treatment of cancer, etc.
  • the present invention proposes a use of the aforementioned chimeric polypeptide, the aforementioned first nucleic acid molecule, the aforementioned first expression vector, the aforementioned recombinant cell or the aforementioned pharmaceutical composition in the preparation of a drug, and the drug is used to prevent and/or treat a disease.
  • the cell expressing the above chimeric polypeptide can recognize the first molecule, which can achieve a variety of different signal output types after binding to the first molecule, such as activation and inhibition of expression of a specific gene, or the immune cell expressing the chimeric polypeptide can secrete a factor (such as a single-chain antibody, cytokine, etc.) with a therapeutic effect after contacting the first molecule, which is used for anti-tumor; and the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in a cell (such as an immune cell), the aforementioned recombinant cell can express the chimeric polypeptide, and the pharmaceutical composition comprises the aforementioned chimeric polypeptide, the first nucleic acid molecule, the first expression vector or the recombinant cell.
  • a factor such as a single-chain antibody, cytokine, etc.
  • a drug containing a chimeric polypeptide, a first nucleic acid molecule, a first expression vector, a recombinant cell or a pharmaceutical composition can target the first molecule for the prevention and/or treatment of related diseases, such as for the treatment of cancer, etc.
  • the present invention proposes a chimeric polypeptide, a first nucleic acid molecule, a first expression vector, a recombinant cell or a pharmaceutical composition for preventing and/or treating a disease.
  • the cell expressing the chimeric polypeptide can recognize the first molecule, and after binding to the first molecule, it can realize a variety of different signal output types, such as activation and inhibition of expression of a specific gene, or the immune cell expressing the chimeric polypeptide can secrete a factor (such as a single-chain antibody, a cytokine, etc.) with a therapeutic effect after contacting the first molecule, which is used for anti-tumor; and the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in a cell (such as an immune cell), the chimeric polypeptide can be expressed on the recombinant cell, and the pharmaceutical composition comprises the chimeric polypeptide, the first nucleic acid molecule
  • a drug containing a chimeric polypeptide, a first nucleic acid molecule, a first expression vector, a recombinant cell or a pharmaceutical composition can target the first molecule for preventing and/or treating related diseases, such as cancer.
  • the present invention proposes a method for treating or preventing a disease.
  • the method comprises: administering a pharmaceutically acceptable amount of the aforementioned recombinant cell or the aforementioned pharmaceutical composition to a subject.
  • the cell expressing the above-mentioned chimeric polypeptide can recognize the first molecule, which can achieve a variety of different signal output types after binding to the first molecule, such as activation and inhibition of expression of a specific gene, or the immune cell expressing the chimeric polypeptide can secrete a factor with a therapeutic effect (such as a single-chain antibody, cytokine, etc.) after contacting the first molecule, for anti-tumor; and, the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in a cell (such as an immune cell), the aforementioned recombinant cell can express the chimeric polypeptide, and the pharmaceutical composition comprises the aforementioned chimeric polypeptide, the first nucleic acid molecule, the first expression vector or the recombinant cell.
  • the drug of the recombinant cell or pharmaceutical composition containing the chimeric polypeptide can target the first molecule for preventing and/or treating related
  • the present invention provides a method for activating immune cells.
  • the method comprises: first contacting the immune cell with a first molecule, wherein the immune cell expresses the aforementioned chimeric polypeptide.
  • the immune cell expressing the aforementioned chimeric polypeptide can recognize the first molecule, and after binding to the first molecule, the immune cell can be activated.
  • the present invention proposes a method for tracking the contact between a first cell and a second cell.
  • the method includes: bringing the first cell and the second cell into second contact, wherein the first cell expresses the aforementioned chimeric polypeptide and the reporter protein, the intracellular region of the chimeric polypeptide is a transcription activator protein, the 5' end of the third nucleic acid molecule encoding the reporter protein is connected to the inducible expression nucleic acid sequence and the inducible expression nucleic acid sequence, the inducible expression nucleic acid sequence is used to bind to the transcription activator protein, and the second cell expresses the first molecule; based on the detection result of the reporter protein in the first cell, determine the contact between the first cell and the second cell.
  • the above method can be used to determine whether the first cell and the second cell are in contact based on the detection result of the reporter protein.
  • FIG1 is a schematic diagram of the structure of the SynNotch synthetic receptor in Example 1 of the present invention.
  • FIG2 is a schematic diagram of SynNotch synthetic receptor cells recognizing specific extracellular signals in Example 2 of the present invention.
  • FIG3 is a schematic diagram of the structure of membrane-anchored GFP in Example 2 of the present invention.
  • FIG5 is a schematic diagram of the structure of membrane-anchored CD19 in Example 3 of the present invention.
  • Figure 6 shows the activation results of SynNotch-CAR-Jurkat T cells by target cancer cells in Example 3 of the present invention
  • Figure 7 shows the killing efficiency of SynNotch-CAR-NK 92 cells on target cancer cells in Example 4 of the present invention.
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as “first” and “second” may explicitly or implicitly include one, two or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality” is two or more.
  • the terms “optionally”, “optional” or “optionally” generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
  • the terms “identity”, “homology” or “similarity” are used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Day Hoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al.
  • GAP Genetics Computing Group
  • the term "at least 80% identity” refers to at least 80%, and may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to each reference sequence.
  • the term "expression vector” generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicate itself, and transfers the inserted nucleic acid molecule into the host cell and/or between host cells.
  • the expression vector may include a vector mainly used to insert DNA or RNA into a cell, a vector mainly used to replicate DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA.
  • the expression vector also includes vectors with multiple of the above functions.
  • the expression vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Usually, by culturing a cell containing the expression vector The expression vector can produce the desired expression product.
  • the term “recombinant cell” generally refers to a cell that has unique traits of stable inheritance by modifying or recombining the genetic material of a host cell using genetic engineering technology or cell fusion technology.
  • the term “host cell” refers to a prokaryotic cell or eukaryotic cell that can be introduced into a recombinant expression vector.
  • the terms "transformed” or “transfected” used herein refer to the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention and can be used for the expression and/or secretion of a target protein.
  • the term "pharmaceutical composition” generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Typically, the composition is prepared by uniformly and thoroughly combining the active chimeric polypeptide or recombinant cell with a liquid carrier, a finely divided solid carrier, or both.
  • the term "pharmaceutically acceptable excipient” may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients that are incompatible with the chimeric polypeptide or recombinant cell of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also contemplated by the present invention.
  • the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means.
  • the recombinant cell or pharmaceutical composition of the present invention can be administered by any common route as long as it can reach the desired tissue.
  • Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified modes of administration.
  • the composition of the present invention is administered by intravenous or subcutaneous injection.
  • treatment refers to the use of drugs to obtain a desired pharmacological and/or physiological effect.
  • the effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and/or may be therapeutic in terms of partially or completely curing a disease and/or the adverse effects caused by the disease.
  • Treatment covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease.
  • Treatment covers any medication that administers a chimeric polypeptide, recombinant cell, pharmaceutical composition or drug to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a chimeric polypeptide, recombinant cell or pharmaceutical composition described herein to an individual in need.
  • the present invention proposes a use of an isolated polypeptide in preparing a SynNotch synthetic receptor, a chimeric polypeptide, a first nucleic acid molecule, a first expression vector, a recombinant cell, a pharmaceutical composition and its use, a method for activating immune cells, and a method for tracking first cell-second cell contact, which will be described in detail below.
  • the present invention provides a use of an isolated polypeptide in preparing a SynNotch synthetic receptor, wherein the isolated polypeptide has at least 80% identity with the transmembrane region of the Notch receptor protein from Xenopus tropicalis.
  • the inventors have found that the SynNotch synthetic receptor prepared by selecting the transmembrane region of the Notch receptor protein from Xenopus tropicalis can improve its ability and efficiency of downstream gene transcriptional activation, and reduce the background leakage of the SynNotch synthetic receptor.
  • the isolated polypeptide has 100% identity with the transmembrane region of the Notch receptor protein derived from Xenopus tropicalis.
  • the transmembrane region of the Notch receptor protein derived from Xenopus tropicalis has an amino acid sequence as shown in SEQ ID NO:1.
  • the present invention provides a chimeric polypeptide.
  • the chimeric polypeptide comprises: an extracellular region, the extracellular region has the activity of binding to a first molecule; a transmembrane region, the N-terminus of the transmembrane region is connected to the C-terminus of the extracellular region; an intracellular region, the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region; wherein the transmembrane region comprises an isolated polypeptide, and the isolated polypeptide has at least 80% identity with the transmembrane region of the Notch receptor protein derived from tropical clawed frog.
  • the inventors have found that selecting the transmembrane region of the Notch receptor protein of tropical clawed frog as the transmembrane region of the chimeric polypeptide (i.e., the SynNotch synthetic receptor) can improve the ability and efficiency of the chimeric polypeptide to activate downstream gene transcription and reduce the background leakage of the chimeric polypeptide.
  • the cells expressing the chimeric polypeptide can recognize
  • the first molecule can realize a variety of different signal output types after binding to the first molecule, such as activation and inhibition of expression of specific genes, and is used to prepare immune cells expressing chimeric polypeptides, which can secrete factors with therapeutic effects (such as single-chain antibodies, cytokines, etc.) for anti-tumor after contacting the first molecule.
  • the isolated polypeptide has 100% identity with the transmembrane region of the Notch receptor protein derived from Xenopus tropicalis.
  • the transmembrane region of the Notch receptor protein derived from Xenopus tropicalis has an amino acid sequence as shown in SEQ ID NO:1.
  • the transmembrane region further includes an epidermal growth factor-like repeat sequence (EGF repeat) and/or a RAM sequence.
  • EGF repeat epidermal growth factor-like repeat sequence
  • the epidermal growth factor-like repeat sequence is an amino acid sequence as shown in SEQ ID NO:15.
  • the RAM sequence is the amino acid sequence shown in SEQ ID NO:16.
  • the C-terminus of the epidermal growth factor-like repeat sequence is connected to the N-terminus of the isolated polypeptide; and/or, the C-terminus of the isolated polypeptide is connected to the N-terminus of the RAM sequence.
  • the first molecule includes at least one of a tumor antigen, a virus, a bacterium, an endotoxin, an antibody, a cell receptor and a ligand of a cell receptor.
  • tumor antigen generally refers to an antigenic substance that appears or is overexpressed during the occurrence and development of a tumor. According to the classification method of tumor antigen specificity, it is divided into tumor-specific antigens and tumor-associated antigens.
  • TSA tumor-specific antigens
  • TAA tumor-associated antigens
  • TAA tumor-associated antigens
  • TAA refer to antigens that are unique to non-tumor cells and also exist in normal cells and other tissues, but their content increases significantly when the cells become cancerous.
  • they include but are not limited to PD-L1, PD-1, TGF- ⁇ , CEA, GD2 and GD3.
  • cell receptor or “receptor” should be understood in a broad sense, and may refer to molecules located on the cell membrane that can recognize and bind to various signal molecules (ligands) outside the cell, including but not limited to growth factor receptors (such as VEGF receptors), (NKG2D polypeptides (receptors for MICA, MICB and ULB6), cytokine receptors (such as IL-13 receptors, IL-2 receptors, etc.), epidermal growth factor (EGF) receptors, Her2, CD27, natural cytotoxicity receptors (NCRs) (such as NKP30 (NCR3/CD337) polypeptides (receptors for HLA-B associated transcript 3 (BAT3) and B7-H6), etc.), T cell antigen receptors, dihydrofolate receptors, chimeric cytokine receptors, Fc receptors, extracellular matrix receptors (such as integrins), cell adhesion receptors (such as cadherins), immunomodul
  • growth factor receptors such
  • ligand of cell receptor should be understood in a broad sense and may refer to chemical substances that can bind to and interact with cell membrane receptors and produce specific biological effects, such as polypeptides, nucleic acids, glycoproteins, small molecules, carbohydrates, lipids, glycolipids, lipoproteins, lipopolysaccharides, etc., including but not limited to cytokines (e.g., IL-13, etc.), growth factors (e.g., heregulin, vascular endothelial growth factor (VEGF)), etc.), peptide hormones, integrin binding peptides (e.g., peptides including the sequence Arg-Gly-Asp), N-glycans, etc.
  • cytokines e.g., IL-13, etc.
  • growth factors e.g., heregulin, vascular endothelial growth factor (VEGF)
  • VEGF vascular endothelial growth factor
  • peptide hormones e.g.,
  • the ligand is VEGF and the receptor is VEGF receptor; or the ligand is heregulin and the receptor is Her2.
  • cytokine should be understood in a broad sense, and may refer to a class of proteins or small molecule polypeptides that can transmit information between cells and have immune regulation and effector functions, such as IL-10.
  • Cytokine receptor should be understood in a broad sense, and may refer to receptors on the cell surface that can bind to cytokines, such as Her2 and IL-10R.
  • the tumor antigen is a tumor-specific antigen.
  • the first molecule includes GFP, eGFP, CD19, ALPPL2, BCMA, SIRP ⁇ , CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44, CD44v6, CD45, CD48, CD51, CD52, CD56, CD59, CD66, CD70, CD71, CD72, CD73, CD74, CD79A, CD79 B.
  • the extracellular region includes a first binding protein or a fragment thereof that binds to the first molecule
  • the first binding protein or fragment thereof includes at least one of an antibody or a functional fragment thereof, a receptor, a ligand of a receptor, and a cell adhesion molecule.
  • cell adhesion molecule may refer to a polypeptide that binds to a component of the extracellular matrix (ECM) or to a cell surface molecule.
  • the cell adhesion molecule may be the extracellular region of a CAM, wherein the CAM may be a calcium-independent adhesion molecule, such as a CAM that is an immunoglobulin superfamily CAM, or a calcium-dependent adhesion molecule, such as a CAM that is an integrin, a cadherin, or a selectin;
  • the cell adhesion molecule may be an integrin, such as a cadherin, such as E-cadherin, P-cadherin, N-cadherin, R-cadherin, M-cadherin, etc.;
  • the cell adhesion molecule may also be a selectin, such as E-selectin, L-selectin, or P-selectin.
  • the term "antibody” is used in the broadest sense, which can include full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, and the specific structure is not limited as long as they show the desired biological activity. It usually includes a light chain with a lighter molecular weight and a heavy chain with a heavier molecular weight, and the heavy chain (H chain) and the light chain (L chain) are connected by a disulfide bond to form an antibody molecule.
  • the amino acid sequence of the amino terminal (N terminal) of the peptide chain varies greatly, which is called the variable region (V region); the carboxyl terminal (C terminal) is relatively stable and varies little, which is called the constant region (C region).
  • the V regions of the L chain and the H chain are called VL and VH, respectively.
  • full-length antibody As used herein, the terms “full-length antibody”, “full-length monoclonal antibody” or “full-length monoclonal antibody” are composed of at least two identical light chains and at least two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) or immunoglobulin E (IgE).
  • IgG immunoglobulin G
  • IgA immunoglobulin A
  • IgM immunoglobulin M
  • IgD immunoglobulin D
  • IgE immunoglobulin E
  • the term "functional fragment” is a fragment comprising part or all of an antibody, which lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen, for example, the fragment may comprise part or all of an antibody CDR.
  • Such fragments are biologically active because they bind to an antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope.
  • Such fragments are selected from Fab, Fv, scFv or single domain antibodies.
  • Such fragments can be produced by recombinant nucleic acid technology, or can be produced by enzymatic cleavage or chemical cleavage of antigen-binding molecules (including intact antibodies).
  • the first binding protein or fragment thereof is a monoclonal antibody or a polyclonal antibody that binds to the first molecule.
  • polyantibody and “multispecific antibody” are synonymous, both referring to antibodies that can recognize multiple antigenic epitopes, such as antibodies that can recognize two antigenic epitopes (bispecific antibodies, referred to as bispecific antibodies), antibodies that recognize three antigenic epitopes, or antibodies that recognize four antigenic epitopes. They are understood in a broad sense, and the specific structure is not limited, as long as they can recognize multiple antigenic epitopes. In the present invention, at least one of the multiple antigenic epitopes is derived from the first molecule.
  • the monoclonal antibody includes at least one of a Fab antibody, a F(ab') 2 fragment, a Fv antibody, a single-chain antibody, a single-domain antibody and a minimum recognition unit.
  • single domain antibody single domain antibody
  • nonantibody single domain antibody
  • VHH antibody single domain antibody
  • Single domain antibody single domain antibody
  • N heavy chain antibody
  • VH heavy chain variable region
  • conventional CH2 and CH3 regions which specifically bind to antigen proteins (e.g., D-dimers) through the heavy chain variable region.
  • Fab antibody or “Fab fragment” generally refers to an antibody or fragment containing only the Fab molecule, which is composed of the VH and CH1 of the heavy chain and a complete light chain, with the light chain and the heavy chain connected by a disulfide bond.
  • F(ab') 2 antibody or "F(ab') 2 fragment” has two antigen-binding F(ab') portions linked together by disulfide bonds.
  • Fv antibody or “Fv fragment” generally refers to an antibody or fragment consisting only of a light chain variable region (VL) and a heavy chain variable region (VH) connected by non-covalent bonds, and is the smallest functional fragment of an antibody that retains a complete antigen binding site.
  • VL light chain variable region
  • VH heavy chain variable region
  • single-chain antibody and “scFv fragment” refer to antibodies or fragments formed by connecting the heavy chain variable region and the light chain variable region of an antibody via a short peptide.
  • minimum recognition unit and “MRU” both refer to antibodies or fragments consisting of only one CDR, and their molecular weight is very small, accounting for only about 1% of the complete antibody.
  • the intracellular region includes at least one of a transcription activator protein, a transcription repressor protein, a transcription factor, a site-specific nuclease, a recombinase, an activating immune receptor intracellular domain and an inhibitory immune receptor intracellular domain.
  • the intracellular region includes at least one of GaL4 VP64, GaL4 VP16, tetR VP64, ZFHD1 VP64, Gal4 KRAB, HAP1 VP16, LexA-VP64, Cas9 and Cas13.
  • the Gal4 VP64 has an amino acid sequence as shown in SEQ ID NO:2.
  • the transmembrane region and the intracellular region have an amino acid sequence as shown in SEQ ID NO:3.
  • the chimeric polypeptide has an amino acid sequence as shown in SEQ ID NO:4.
  • nucleic acid molecules Nucleic acid molecules, vectors, recombinant cells and pharmaceutical compositions
  • the present invention provides a first nucleic acid molecule.
  • the first nucleic acid molecule encodes the chimeric polypeptide as claimed in claim 1.
  • the first nucleic acid molecule according to an embodiment of the present invention can encode the chimeric polypeptide as described above.
  • the nucleic acid molecule is DNA.
  • the first nucleic acid molecule mentioned herein those skilled in the art will understand that it actually includes any one or two of the complementary double strands. For convenience, in this article, although only one strand is provided in most cases, the other strand complementary thereto is actually disclosed.
  • the molecular sequence in the present invention includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.
  • the present invention provides a first expression vector.
  • the first expression vector carries the aforementioned first nucleic acid molecule.
  • the first nucleic acid molecule can be directly or indirectly connected to the control element on the vector, as long as these control elements can control the translation and expression of the first nucleic acid molecule.
  • these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself.
  • the first nucleic acid molecule can be operably connected to the control element.
  • operably linked means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended functions of regulating the transcription and translation of the exogenous gene.
  • control elements in the vector such as transcription control sequences and translation control sequences, etc.
  • Commonly used vectors can be, for example, plasmids, bacteriophages, etc. After the vectors according to some specific embodiments of the present invention are introduced into suitable receiving cells (also called receptor cells or host cells), the expression of the aforementioned chimeric polypeptide can be effectively achieved under the mediation of the regulatory system.
  • the first expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.
  • the first expression vector is a plasmid expression vector.
  • the present invention provides a recombinant cell.
  • the recombinant cell includes: carrying the aforementioned first nucleic acid molecule or the aforementioned first expression vector; or, expressing the aforementioned chimeric polypeptide.
  • the recombinant cell can effectively express the aforementioned chimeric polypeptide in the recombinant cell under suitable conditions.
  • suitable conditions refer to conditions suitable for the expression of the aforementioned chimeric polypeptide. It is easy for those skilled in the art to understand that the conditions suitable for the expression of the aforementioned chimeric polypeptide include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell states, suitable cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions” are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the chimeric polypeptide according to the specific environment of the laboratory.
  • the recombinant cell is obtained by introducing the aforementioned first expression vector into a host cell.
  • the host cells include immune cells, neurons, progenitor cells or precursor cells, epithelial cells, endothelial cells and stem cells. At least one of.
  • the host cell includes at least one of T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells and ⁇ T cells.
  • the recombinant cell further comprises: a second nucleic acid molecule encoding a chimeric antigen receptor, an NK cell receptor or a T cell receptor or a second expression vector carrying the second nucleic acid molecule; or expressing the chimeric antigen receptor, T cell receptor or NK cell receptor.
  • a second nucleic acid molecule encoding a chimeric antigen receptor, an NK cell receptor or a T cell receptor or a second expression vector carrying the second nucleic acid molecule; or expressing the chimeric antigen receptor, T cell receptor or NK cell receptor.
  • the inventors have found through experiments that the chimeric polypeptide of the present invention is combined with a chimeric antigen receptor, an NK cell receptor or a T cell receptor to jointly transform immune cells (such as Jurkat cells and NK-92 cells).
  • the immune cells combined with the chimeric polypeptide + CAR/TCR can recognize the corresponding ligands and efficiently activate immune cells (such as Jurkat cells and NK-92 cells) to exercise the function of killing targets (such as tumor cells, etc.).
  • immune cells such as Jurkat cells and NK-92 cells
  • chimeric antigen receptor is a fusion protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a different polypeptide than the extracellular domain, and at least one intracellular domain.
  • CAR Chimeric Antigen Receptor
  • CIR chimeric immune receptor
  • the "extracellular domain capable of binding to an antigen” refers to any oligopeptide or polypeptide capable of binding to an antigen.
  • Extracellular domain refers to any oligopeptide or polypeptide known to function as a domain that transmits signals to activate or inhibit intracellular biological processes.
  • T cell receptor is a molecule that can be found on the surface of T cells, which is responsible for recognizing antigens that can bind to MHC molecules.
  • Naturally occurring TCR heterodimers are composed of alpha ( ⁇ ) and beta ( ⁇ ) chains in about 95% of T cells, while about 5% of T cells have TCRs composed of gamma ( ⁇ ) and delta ( ⁇ ) chains.
  • MHC major histocompatibility complex
  • T cells are able to recognize this antigen peptide-MHC complex on the surface of antigen-presenting cells.
  • the binding of TCR to the antigen peptide-MHC complex leads to the activation of T lymphocytes, and TCR is expressed on T lymphocytes through a series of biochemical reactions mediated by related enzymes, co-receptors and specialized auxiliary molecules.
  • the MHC molecule can be a class I or class II MHC molecule.
  • the complex can be in an antigen presenting cell, such as a dendritic cell, a B cell, or any other cell (such as a K562 cell).
  • HLA human leukocyte antigen system
  • MHC human major histocompatibility complex
  • HLA class I antigens A, B, and C
  • HLA class II antigens DP, DQ, and DR
  • HLA alleles A, B, and C present peptides that are primarily derived from intracellular proteins, e.g., proteins expressed within cells.
  • NK cell receptor refers to a molecule that can be found on the surface of NK cells, and can be divided into inhibitory receptors and activating receptors according to the different functions it mediates.
  • inhibitory receptors are used to recognize specific molecules expressed on the surface of normal cells, transduce killing inhibition signals, and inhibit their own killing function; there are many types of inhibitory receptors, including but not limited to CD161, CLRG1, PD1, TIM3, LAG 3, CD96 and TIGIT.
  • Activating receptors are used to recognize the corresponding ligands on the surface of target cells, transduce activation signals into cells, and exert a killing effect.
  • Activating receptors include but are not limited to NKp30, NKp44, NKp46 and CD16.
  • the intracellular region includes a transcriptional activator protein, and the 5' end of the second nucleic acid molecule encoding the chimeric antigen receptor, NK cell receptor or T cell receptor is connected to an inducible expression nucleic acid sequence, and the inducible expression nucleic acid sequence is used to bind to the transcriptional activator protein.
  • the intracellular region is GaL4 VP64
  • the induced expression nucleic acid sequence is a UAS-minimal-CMV sequence.
  • the UAS-minimal-CMV sequence has a nucleic acid sequence as shown in SEQ ID NO:14.
  • the chimeric antigen receptor, NK cell receptor or T cell receptor has the activity of binding to a second molecule.
  • the chimeric antigen receptor binds to the second molecule mainly through its extracellular region.
  • the second molecule includes at least one of a tumor antigen, a virus, a bacterium, an endotoxin, an antibody, a cell receptor and a ligand of a cell receptor.
  • the second molecule includes GFP, eGFP, CD19, ALPPL2, BCMA, SIRP ⁇ , CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44, CD44v6, CD45, CD48, CD51, CD52, CD56, CD59, CD66, CD70, CD71, CD72, CD73, CD74, CD79A, CD 79B, CD80, CD86, CD94, CD95, CD133, CD134, CD140, CD152, CD154, CD158, CD178, CD181, CD182, CD183, CD200, CD210, CD221, CD246, CD252, CD253, CD261, CD262, CD273, CD274, CD 276, CD279, CD295,
  • PSMA PSMA, MAGEA1, 4-1BB, 5T4, BAFF, CA242, CA-IX, MET, CCR4, CNTO888, FAP, MORAb-009, VEGF-A, VEGFR-1 and VEGFR-2 At least one of .
  • the present invention proposes a pharmaceutical composition.
  • the pharmaceutical composition includes the aforementioned chimeric polypeptide, the aforementioned first nucleic acid molecule, the aforementioned first expression vector or the aforementioned recombinant cell.
  • the cell expressing the above chimeric polypeptide can recognize the first molecule, which can achieve a variety of different signal output types after binding to the first molecule, such as activation and inhibition of expression of a specific gene, or the immune cell expressing the chimeric polypeptide can secrete factors with therapeutic effects (such as single-chain antibodies, cytokines, etc.) after contacting the first molecule, which is used for anti-tumor; and the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in cells (such as immune cells), and the chimeric polypeptide can be expressed on the aforementioned recombinant cells. Therefore, a pharmaceutical composition containing the aforementioned chimeric polypeptide, the first nucleic acid molecule, the first expression vector or the recombinant cell can target the first molecule for the prevention and/or treatment of related diseases, such as cancer.
  • it further includes a pharmaceutically acceptable excipient.
  • the present invention proposes a use of the aforementioned chimeric polypeptide, the aforementioned first nucleic acid molecule, the aforementioned first expression vector, the aforementioned recombinant cell or the aforementioned pharmaceutical composition in the preparation of a drug, and the drug is used to prevent and/or treat a disease.
  • the cell expressing the above-mentioned chimeric polypeptide can recognize the first molecule, which can achieve a variety of different signal output types after binding to the first molecule, such as activation and inhibition of expression of a specific gene, or the immune cell expressing the chimeric polypeptide can secrete a factor (such as a single-chain antibody, cytokine, etc.) with a therapeutic effect after contacting the first molecule, which is used for anti-tumor; and the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in a cell (such as an immune cell), the aforementioned recombinant cell can express the chimeric polypeptide, and the pharmaceutical composition comprises the aforementioned chimeric polypeptide, the first nucleic acid molecule, the first expression vector or the recombinant cell.
  • a factor such as a single-chain antibody, cytokine, etc.
  • a drug containing a chimeric polypeptide, a first nucleic acid molecule, a first expression vector, a recombinant cell or a pharmaceutical composition can target the first molecule for the prevention and/or treatment of related diseases, such as cancer.
  • the present invention proposes a chimeric polypeptide, a first nucleic acid molecule, a first expression vector, a recombinant cell or a pharmaceutical composition for preventing and/or treating a disease.
  • the cell expressing the chimeric polypeptide can recognize the first molecule, and after binding to the first molecule, it can realize a variety of different signal output types, such as activation and inhibition of expression of a specific gene, or the immune cell expressing the chimeric polypeptide can secrete a factor (such as a single-chain antibody, a cytokine, etc.) with a therapeutic effect after contacting the first molecule, which is used for anti-tumor; and the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in a cell (such as an immune cell), the chimeric polypeptide can be expressed on the recombinant cell, and the pharmaceutical composition comprises the chimeric polypeptide, the first nucleic acid molecule
  • a drug containing a chimeric polypeptide, a first nucleic acid molecule, a first expression vector, a recombinant cell or a pharmaceutical composition can target the first molecule for preventing and/or treating related diseases, such as cancer.
  • the above two aspects of use may further include at least one of the following technical features:
  • the disease includes cancer or tumor, and immune-related disease.
  • the disease includes cancer or tumor, autoimmune disease, inflammation and related diseases caused by cell aging.
  • cancer can be any unregulated cell growth.
  • it can be non-small cell lung cancer, papillary thyroid cancer, glioblastoma multiforme, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, bile duct cancer or sarcoma, acute myeloid leukemia, large cell neuroendocrine carcinoma, neuroblastoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, cervical cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma or gastric cancer, etc.
  • the present invention provides a method for activating immune cells.
  • the method comprises: first contacting the immune cell with a first molecule, wherein the immune cell expresses the aforementioned chimeric polypeptide.
  • the immune cell expressing the aforementioned chimeric polypeptide can recognize the first molecule, which can activate the immune cell after binding to the first molecule, and is particularly suitable for scientific research, by culturing the immune cell in vitro to obtain activated immune cells.
  • the intracellular region of the chimeric polypeptide includes a transcriptional activator protein
  • the immune cell expresses a chimeric antigen receptor, an NK cell receptor or a T cell receptor
  • the chimeric antigen receptor, the NK cell receptor or the T cell receptor includes an antibody or a functional fragment thereof that binds to a predetermined antigen
  • the 5' end of the second nucleic acid molecule encoding the chimeric antigen receptor, the NK cell receptor or the T cell receptor is connected to an inducible expression nucleic acid sequence, and the inducible expression nucleic acid sequence is used to bind to the transcriptional activator protein
  • the method further includes: after the first contact, the immune cell releases the transcriptional activator protein and expresses the chimeric antigen receptor, the NK cell receptor or the T cell receptor; the chimeric antigen receptor, the NK cell receptor or the T cell receptor of the immune cell is combined with the predetermined antigen to activate the immune cell.
  • the induced expression nucleic acid sequence has a nucleic acid sequence as shown in SEQ ID NO:14.
  • the first molecule is a tumor antigen.
  • the first molecule includes GFP, eGFP, CD19, ALPPL2, BCMA, SIRP ⁇ , CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44, CD44v6, CD45, CD48, CD51, CD52, CD56, CD59, CD66, CD70, CD71, CD72, CD73, CD74, CD79A, CD79B, CD80, CD86, CD94, CD95, CD133, CD134, CD140, CD152, and CD156.
  • the immune cells include at least one of T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells and ⁇ T cells.
  • the present invention proposes a method for tracking the contact between a first cell and a second cell.
  • the method includes: bringing the first cell and the second cell into second contact, wherein the first cell expresses the aforementioned chimeric polypeptide and the reporter protein, the intracellular region of the chimeric polypeptide is a transcription activator protein, the 5' end of the third nucleic acid molecule encoding the reporter protein is connected to an inducible expression nucleic acid sequence, the inducible expression nucleic acid sequence is used to bind to the transcription activator protein, and the second cell expresses the first molecule; based on the detection result of the reporter protein in the first cell, determining the contact between the first cell and the second cell.
  • the above method can be used to determine whether the first cell and the second cell are in contact based on the detection result of the reporter protein, which is particularly suitable for scientific research, and can be used to determine whether the two cells are in contact in vitro.
  • the induced expression nucleic acid sequence has a nucleic acid sequence as shown in SEQ ID NO:14.
  • the first molecule is on the surface of the second cell, is immobilized on an insoluble substrate, exists in an extracellular matrix, exists in an artificial matrix, or is soluble.
  • the first molecule is a tumor antigen.
  • the first molecule includes GFP, eGFP, CD19, ALPPL2, BCMA, SIRP ⁇ , CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44, CD44v6, CD45, CD48, CD51, CD52, CD56, CD59, CD66, CD70, CD71, CD72, CD73, CD74, CD79A, CD79B, CD80, CD86, CD94, CD95, CD133, CD134, CD140, CD152, and CD156.
  • the first cell or the second cell includes at least one of an immune cell, a neuron, a progenitor cell or a precursor cell, an epithelial cell, an endothelial cell and a stem cell.
  • the first cell includes at least one of T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells and ⁇ T cells.
  • the present invention provides a method for regulating cell activity.
  • the method comprises: contacting the cell with a first molecule for a third time, wherein the cell expresses the aforementioned chimeric polypeptide.
  • the cell comprises at least one of an immune cell, a neuron, a progenitor cell or a precursor cell, an epithelial cell, an endothelial cell and a stem cell.
  • the cells include at least one of T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells and ⁇ T cells.
  • the present invention proposes a method for promoting the expression of genes or proteins in cells.
  • the method comprises: contacting the cell with a first molecule for the fourth time, wherein the cell expresses the aforementioned chimeric polypeptide, the cell carries the gene or expresses the protein, wherein the gene comprises a nucleic acid molecule encoding at least one of a chimeric antigen receptor, a second chimeric polypeptide, a translation regulator, a cytokine, a hormone, a chemokine, an antibody, and a protein located in the intracellular region, or the protein comprises at least one of a chimeric antigen receptor, a second chimeric polypeptide, a translation regulator, a cytokine, a hormone, a chemokine, an antibody, and a protein located in the intracellular region.
  • the intracellular region of the chimeric polypeptide is a transcription activator protein, and the chimeric antigen receptor, the second chimeric polypeptide, the translation regulator,
  • the cytokine, hormone, chemokine or antibody comprises a second binding protein of the transcriptional activator protein or a fragment thereof.
  • the cell comprises at least one of an immune cell, a neuron, a progenitor cell or a precursor cell, an epithelial cell, an endothelial cell and a stem cell.
  • the cells include at least one of T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells and ⁇ T cells.
  • the present invention proposes a method for treating or preventing a disease.
  • the method comprises: administering a pharmaceutically acceptable amount of the aforementioned recombinant cell or the aforementioned pharmaceutical composition to a subject.
  • the cell expressing the above-mentioned chimeric polypeptide can recognize the first molecule, which can achieve a variety of different signal output types after binding to the first molecule, such as activation and inhibition of expression of a specific gene, or the immune cell expressing the chimeric polypeptide can secrete a factor with a therapeutic effect (such as a single-chain antibody, cytokine, etc.) after contacting the first molecule, for anti-tumor; and, the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in a cell (such as an immune cell), the aforementioned recombinant cell can express the chimeric polypeptide, and the pharmaceutical composition comprises the aforementioned chimeric polypeptide, the first nucleic acid molecule, the first expression vector or the recombinant cell.
  • the drug of the recombinant cell or pharmaceutical composition containing the chimeric polypeptide can target the first molecule for preventing and/or treating related
  • the effective amount of the recombinant protein or pharmaceutical composition of the present invention may vary depending on the mode of administration and the severity of the disease to be treated.
  • the selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials).
  • the factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, several divided doses may be administered daily, or the dose may be reduced proportionally, depending on the urgency of the treatment situation.
  • the recombinant protein or pharmaceutical composition of the present invention can be incorporated into a drug suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • parenteral administration e.g., intravenous, subcutaneous, intraperitoneal, intramuscular
  • these drugs can be prepared in various forms.
  • liquid, semisolid and solid dosage forms including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders.
  • Typical drugs are in the form of injection solutions or infusion solutions.
  • the aforementioned recombinant protein or pharmaceutical composition can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.
  • the administration route of the method is subcutaneous injection or intravenous injection.
  • the disease includes cancer or tumor, immune-related disease.
  • the disease includes cancer or tumor, autoimmune disease, inflammation and related diseases caused by cell senescence.
  • the tumor or cancer includes but is not limited to non-small cell lung cancer, papillary thyroid cancer, glioblastoma multiforme, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, bile duct cancer or sarcoma, acute myeloid leukemia, large cell neuroendocrine carcinoma, neuroblastoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, cervical cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma or gastric cancer, etc.
  • Example 1 Design of SynNotch synthetic receptor and construction of its expression plasmid vector
  • the structure of the SynNotch synthetic receptor (referred to as the SynNotch receptor) includes an extracellular domain (also known as an extracellular region), a Notch transmembrane domain (also known as the transmembrane region of the Notch receptor protein, referred to as the transmembrane region) and an intracellular domain (also known as the intracellular region), wherein the extracellular domain can be replaced by a single-chain antibody or a nanobody, etc., and the intracellular domain can be replaced by a transcriptional activation or transcriptional inhibition domain, etc.
  • an extracellular domain also known as an extracellular region
  • a Notch transmembrane domain also known as the transmembrane region of the Notch receptor protein
  • an intracellular domain also known as the intracellular region
  • the present invention uses an anti-GFP single-chain antibody as the extracellular domain portion, GAL4-VP64 as the intracellular domain portion, and a mouse or tropical clawed frog or Notch transmembrane domain that can be recognized and cleaved by a protease as the Notch transmembrane domain to obtain a modified SynNotch receptor.
  • the structure of the SynNotch receptor designed and modified by the present invention is shown in Figure 1.
  • Figure 1 shows the structure of a SynNotch receptor containing a Notch transmembrane domain (Notch core) of a mouse or tropical clawed frog.
  • SynNotch synthetic receptor 1 The synthetic SynNotch receptor containing the tropical clawed frog Notch transmembrane domain (referred to as SynNotch synthetic receptor 1) has the amino acid sequence shown in SEQ ID NO:4 and the nucleotide sequence shown in SEQ ID NO:5, and the synthetic SynNotch receptor containing the mouse Notch transmembrane domain (referred to as SynNotch synthetic receptor 2) has the amino acid sequence shown in SEQ ID NO:6 and the nucleotide sequence shown in SEQ ID NO:7, and then GENEWIZ synthesizes the nucleotide sequence shown in SEQ ID NO:5 and the nucleotide sequence shown in SEQ ID NO:7 into the Puc57 vector respectively;
  • the pCDH-EV vector was double-digested with EocR1 and BamH1 endonucleases, and the double-digested vector was recovered by gel electrophoresis using 1% agarose gel for 20 min;
  • nucleotide sequence shown in SEQ ID NO:5 and the nucleotide sequence shown in SEQ ID NO:7 were obtained from the two pUC57 vectors in step (1) by PCR, and then the nucleotide sequence shown in SEQ ID NO:5 and the nucleotide sequence shown in SEQ ID NO:7 were homologously recombined with the double-enzyme-cut vector obtained in step (2) and the nucleotide fragment of the SynNotch synthetic receptor, respectively, to obtain two expression plasmid vectors, pCDH-antiGFP-XENTR-SynNotch-Gal4-VP64 and pCDH-antiGFP-MOUSE-SynNotch-Gal4-VP64.
  • the specific process of SynNotch receptor cells recognizing specific extracellular signals is as follows: when the anti-GFP antibody single chain of the extracellular domain of the SynNotch receptor binds to the GFP antigen of the sender cell, the Notch transmembrane domain is cleaved by protease, GAL4VP64 is released into the cell, GAL4 can specifically recognize and bind to the GAL4 binding site (UAS region), and the VP64 transcriptional activation element will activate the transcription of the downstream BFP gene and promote the expression of the BFP fluorescent protein. See Figure 2 for details.
  • the present invention uses HT1080 cells as sender cells and another batch of HT1080 cells as receiver cells to verify the ability of the expression plasmid vector (pCDH-antiGFP-XENTR-SynNotch-Gal4VP64) prepared in Example 1 to enhance the transcriptional activation of downstream genes at the cellular level.
  • the specific verification steps are as follows:
  • HT1080 cell line infected with lentivirus can stably express membrane-anchored GFP and serve as a delivery cell (the structure of membrane-anchored GFP is shown in FIG3 , and GFP has an amino acid sequence as shown in SEQ ID NO: 8 and a nucleotide sequence as shown in SEQ ID NO: 9).
  • amino acid and nucleotide sequences of GFP are as follows:
  • HT1080 cells were taken as receiving cells, first infected with pHR_Gal4UAS_tBFP_PGK_mCherry lentivirus (purchased from Addgene, #79130, after the lentivirus infects the host cells, it can be induced by GAL4VP64 to express BFP fluorescent protein), sorted out cells that can stably and highly express pHR_Gal4UAS_tBFP_PGK_mCherry, and then infected with pCDH-antiGFP-XENTR-SynNotch-Gal4VP64 lentivirus.
  • pHR_Gal4UAS_tBFP_PGK_mCherry lentivirus purchased from Addgene, #79130, after the lentivirus infects the host cells, it can be induced by GAL4VP64 to express BFP fluorescent protein
  • pHR_Gal4UAS_tBFP_PGK_mCherry and pCDH-antiGFP-XENTR-SynNotch-Gal4VP64 lentiviruses are prepared by co-transfecting the expression plasmid (pCDH-antiGFP-XENTR-SynNotch-Gal4VP64 or pHR_Gal4UAS_tBFP_PGK_mCherry), pMD2.G and psPAX2 into a 15 cm culture dish containing HEK293-T cells at a density of 90%. Three days later, the cell supernatant was collected, the virus solution was filtered using a 0.45 ⁇ M filter, and the virus was concentrated.
  • Figure 4A is a comparison of fluorescence microscope photos, where MOUSE-Syn represents a synthetic SynNotch receptor containing a mouse Notch transmembrane domain (referred to as SynNotch synthetic receptor 2), which is the original system; XENTR-Syn represents a synthetic SynNotch receptor containing a tropical clawed frog Notch transmembrane domain (referred to as SynNotch synthetic receptor 1). As shown in Figure 4A, compared with SynNotch synthetic receptor 2, SynNotch synthetic receptor 1 can significantly improve the efficiency of activation.
  • Figure 4B is the average fluorescence intensity of BFP blue fluorescent protein expressed in HT1080 cells analyzed by ImageJ software. As shown in Figure 4B, compared with SynNotch synthetic receptor 2 (i.e., MOUSE group), the activation efficiency of SynNotch synthetic receptor 1 (i.e., XENTR group) was increased by 95.1%, and the leakage level of SynNotch synthetic receptor 1 (i.e., XENTR group) was reduced by 56.4%.
  • SynNotch synthetic receptor 2 i.e., MOUSE group
  • the transmitting cells K562 and the receiving cells Jurkat were co-cultured for 48 hours to observe whether the contact between the suspended cells and the suspended cells can activate the SynNotch system; at the same time, the transmitting cells HT1080 and the receiving cells Jurkat were co-cultured for 48 hours to observe whether the contact between the suspended cells and the adherent cells can activate the SynNotch system.
  • the results are shown in Figure 4C and Figure 4D.
  • XENTR-SynNotch and MOUSE-SynNotch can be effectively activated in both the contact between suspended receiving cells and suspended transmitting cells and the contact between suspended receiving cells and attached transmitting cells.
  • the leaky activation efficiency of XENTR-SynNotch is much lower than that of MOUSE-SynNotch, and the effective activation efficiency is slightly higher than that of MOUSE-SynNotch.
  • the SynNotch system designed by the present invention containing the transmembrane region of the Notch receptor protein derived from tropical clawed frog (Xenopus tropicalis) has a stronger activation efficiency than the original SynNotch receptor system containing the transmembrane region of the Notch receptor protein derived from mouse.
  • Example 3 Activation of Jurkat cells by target cancer cells combined with SynNotch-CAR gated chimeric antigen receptor
  • CARs chimeric antigen receptors
  • B-cell cancers a major problem with CAR-T cell cancer immunotherapy is off-target effects, in which therapeutic CAR-T cells damage normal tissues, leading to serious side effects or even death.
  • a possible strategy to alleviate such problems is to give therapeutic CAR-T cells a more precise tumor recognition target, and adding a tumor antigen recognition to enable CAR expression to provide a more precise T cell response is an effective means.
  • SynNotch synthetic receptors can be used in the design of therapeutic CAR-T cells, that is, first detecting the tumor by binding to a tumor-specific cell surface antigen and triggering the expression of CAR only in response to a second tumor-specific antigen in the tumor, thereby This provides both dual antigen control of CAR-T cell activity and a tumor-localized response.
  • SynNotch-CAR-Jurkat T cells chimeric antigen receptors
  • the SynNotch synthetic receptor 1 in Example 2 is packaged with a lentivirus, and the lentivirus packaging can recognize the CAR molecule of CD19.
  • the 5' end of the nucleic acid encoding the CAR molecule includes a UAS-minimal-CMV sequence, which can bind to the intracellular polypeptide produced after the activation of the SynNotch synthetic receptor to induce the expression of the CAR molecule (directly synthesized and lentivirus packaged by GENEWIZ, the CAR molecule has an amino acid sequence as shown in SEQ ID NO: 10 and a nucleotide sequence as shown in SEQ ID NO: 11, and the CAR molecule is hereinafter referred to as an inducible CAR molecule).
  • the above two viruses are used to infect Jurkat T cells at the same time, and Jurkat T cells that stably express the above SynNotch synthetic receptor and inducible CAR molecules are obtained by screening;
  • CD19 has an amino acid sequence as shown in SEQ ID NO: 12 and a nucleotide sequence as shown in SEQ ID NO: 13), and co-expressing membrane-anchored GFP and CD19 as transfer cells;
  • step (2) The three types of K562 cells in step (2) were co-cultured with the Jurkat cells constructed in step (1), and the expression of CD69 and the secretion of IL-2 and TNF- ⁇ of Jurkat cells in each group were detected after 24 hours.
  • the results are shown in Figure 6. Among them, the expression of CD69 and the secretion of IL-2 and TNF- ⁇ represent the activation of Jurkat T cells.
  • SynNotch-CAR-Jurkat T cells can be activated only when exposed to K562 expressing two antigens (GFP and CD19).
  • Example 4 Killing of target cancer cells by NK-92 cells combined with SynNotch-CAR gated chimeric antigen receptor (referred to as SynNotch-CAR-NK-92 cells)
  • the SynNotch synthetic receptor 1 of the present invention can not only play a role in T cells, but should also be universal in other types of immune cells.
  • the SynNotch synthetic receptor (SynNotch synthetic receptor 1 or SynNotch synthetic receptor 2) in Example 2 is packaged with lentivirus, and the lentivirus packaging can recognize the CAR molecule of CD19.
  • the 5' front end of the nucleic acid sequence encoding the CAR molecule includes a UAS-minimal-CMV sequence, which can bind to the intracellular polypeptide produced after the activation of the SynNotch synthetic receptor to induce the expression of the CAR molecule (directly synthesized and lentivirus packaged by GENEWIZ, the CAR molecule has an amino acid sequence as shown in SEQ ID NO: 10 and a nucleotide sequence as shown in SEQ ID NO: 11).
  • NK-92 cells that stably express the above SynNotch synthetic receptor and CAR molecules are obtained by screening; at the same time, NK-92 cells (i.e., CAR-NK) that stably express the CAR molecule that can recognize CD19 are set as the positive killing control group, and NK-EV (NK cells infected with viruses produced by blank plasmids) are set as the negative killing control group;
  • the three types of Huh7 cells in step (3) were co-incubated with the NK-92 cells constructed in (1), and the cells were collected after 48 hours.
  • the dead cells were labeled with PI dye, and the live and dead conditions of the Huh7 cells in each group were analyzed and detected, as shown in Figure 7.
  • the death ratio of Huh7 cells represents the killing of the NK-92 cells on the transfer cells.
  • SynNotch-CAR-NK-92 cells when exposed to Huh7 cells expressing two antigens (GFP and CD19) at the same time, SynNotch-CAR-NK-92 cells can be significantly activated and exert a higher killing effect on Huh7 cells.
  • the killing efficiency of SynNotch-CAR (X-Syn-CAR) containing SynNotch synthetic receptor 1 is slightly improved; and the killing efficiency of NK-92 cells combined with SynNotch-CAR containing SynNotch synthetic receptor 1 is not significantly different from that of NK-92 cells stably expressing CAR molecules (CAR-NK) that can recognize CD19.

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Abstract

本发明提出了一种嵌合多肽。所述嵌合多肽包括：胞外区，所述胞外区具有结合第一分子活性；跨膜区，所述跨膜区的N端与所述胞外区的C端相连；胞内区，所述胞内区的N端与所述跨膜区的C端相连；其中，所述跨膜区包括分离的多肽，所述分离的多肽与来源于热带爪蟾的Notch受体蛋白的跨膜区具有至少80％的同一性。

Description

SynNotch受体及其用途 技术领域
本领域属于生物技术领域，具体地，本发明涉及一种SynNotch受体及其用途，更具体地，本发明涉及一种分离的多肽在制备SynNotch合成受体中的用途、嵌合多肽、第一核酸分子、第一表达载体、重组细胞、药物组合物及其用途、激活免疫细胞的方法、追踪第一细胞-第二细胞接触的方法。
背景技术
随着合成生物学的发展，通过改造细胞使之可以识别细胞外特定的信号，并在改造的细胞中传递识别到的信号，进而做出人工设计的反应已经成为现实，并已经逐渐在生物医疗领域展开了应用。如嵌合抗原受体(CAR)技术在临床治疗中的应用，除此之外，在2016年，Wend ell A.Lim团队基于Notch信号通路开发出了一套SynNotch受体系统，该系统同样可以识别细胞膜表面配体，并且其信号输出并不局限于单纯的免疫细胞激活信号，可以实现多种不同的信号输出类型，如特定基因的表达激活与抑制等。该受体系统可以实现不同输入信号与输出信号的多种组合，且同一体系(细胞)内的多组系统间具有正交性；同时在众多细胞中均可运行，具有普适性；同时具有简易性和可调控性等诸多优秀特征，是应用合成生物学对细胞进行改造的一个强力工具。
生物学中，传统的Notch信号通路(系统)包括Notch对应的配体、Notch分子和Notch调控的下游基因。其中Notch分子是一个跨膜分子，其可以分为胞外结构、跨膜结构域和胞内结构域3个部分。其胞外结构域与对应的Notch配体结合后，配体所在的细胞出现胞吞活动，拉扯Notch分子，使Notch分子的胞外结构域上的S2蛋白酶切割位点暴露出来，被金属蛋白酶ADAMs家族成员等切割；进一步的，伴随着胞外结构域的切割断裂，Notch分子的胞内结构域被γ-分泌酶切割后释放到细胞内，随后入核作为转录因子调控下游相关基因的表达。Wend ell A.Lim团队改造了鼠的Notch1受体，将其胞外域用单链抗体或者纳米抗体替换，胞内域用转录激活或转录抑制的结构域替换，仅保留了可以被蛋白酶识别切割的跨膜结构域，还配备了下游被调控的基因元件。针对不同靶标，胞外域采用特异识别该抗原的抗体单链，而胞内域调控预先设定的目标基因或因子的表达。在同一细胞中可以设计由不同抗原启动的基因调控环路，并具有很好的正交性。SynNotch系统可以被应用到许多细胞类型中，包括神经细胞、肿瘤细胞、表皮细胞和免疫细胞等。CAR技术和SynNotch系统组合应用到免疫细胞改造中，可以实现免疫细胞的“与门”激活，即只有当细胞同时表达两种特定的表面抗原时，才能将免疫细胞激活。改变SynNotch下游调控的基因元件，免疫细胞可以在接触特定抗原后，分泌单链抗体、细胞因子等等具有治疗作用的因子，并且在体外、体内都具有抗肿瘤的作用。可见SynNotch受体系统，在细胞改造中具有极大的技术优势。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题至少之一。为此，本发明提供了一种SynNotch受体，该SynNotch受体可增强其对下游基因的激活能力和减少本底泄露激活水平。
本发明是基于发明人的下列发现而完成的：
SynNotch系统功能十分强大，但是目前常用的初始版本SynNotch系统(又称SynNotch合成受体、SynNotch)具有如下缺点：第一，初始版本SynNotch被激活后，其下游转录表达调控能力并不是很强，即激活下游基因的表达量并不很高，会局限SynNotch的使用，特别是针对需要下游基因有较高的表达量才能发挥效用的情况。第二，初始版本SynNotch的本底泄露水平较高，对于下游需要严谨调控的基因，其较高水平的泄露表达可能会导致SynNotch分子开关的失效，分子表达线路会直接越过SynNotch分子开关实现表达。
然而，发明人经过试验意外发现，上述问题的关键点在于SynNotch分子的跨膜结构域存在不足。初始版本SynNotch合成受体选择了小鼠来源Notch分子的跨膜结构域来作为SynNotch合成受体的跨膜结构域。考虑到不同种属来源的Notch跨膜结构域上所携带的S2和S3切割序列存在差异，以及不同物种来源的S2和S3切割酶存在的构象和序列偏好性，在不同物种的细胞中，使用不同的跨膜结构域所构建的SynNotch合成受体其激活效率和泄露效率必然有所差异。发明人以用SynNotch合成受体改造人源细胞为最终目的，在试验过程中意外发现选择热带爪蟾的Notch跨膜结构域，可以提高SynNotch合成受体对下游基因转录激活的能力和效率，以及降低SynNotch合成受体的本底泄露激活，使SynNotch合成受体能够更加有效的发挥作用。
因此，在本发明的一个方面，本发明提出了一种分离的多肽在制备SynNotch合成受体中的用途，所述分离的多肽与来源于热带爪蟾(Xenopus tropicalis)的Notch受体蛋白的跨膜区具有至少80％的同一性。发明人发现，选择热带爪蟾的Notch受体蛋白的跨膜区制备的SynNotch合成受体，可以提高其对下游基因转录激活的能力和效率，降低SynNotch合成受体的本底泄露。
本发明的另一方面，本发明提出了一种嵌合多肽。根据本发明的实施例，所述嵌合多肽包括：胞外区，所述胞外区具有结合第一分子活性；跨膜区，所述跨膜区的N端与所述胞外区的C端相连；胞内区，所述胞内区的N端与所述跨膜区的C端相连；其中，所述跨膜区包括分离的多肽，所述分离的多肽与来源于热带爪蟾的Notch受体蛋白的跨膜区具有至少80％的同一性。本发明的嵌合多肽选择热带爪蟾的Notch受体蛋白的跨膜区作为嵌合多肽(即为SynNotch合成受体)的跨膜区，可以提高嵌合多肽对下游基因转录激活的能力和效率，以及降低嵌合多肽的本底泄露。
在本发明的又一方面，本发明提出了一种第一核酸分子。根据本发明的实施例，所述第一核酸分子编码权利要求前述的嵌合多肽。根据本发明实施例的第一核酸分子所编码前述的嵌合多肽。
在本发明的又一方面，本发明提出了一种第一表达载体。根据本发明的实施例，所述第一表达载体携带前述的第一核酸分子。本发明的第一表达载体导入合适的接收细胞后，可实现前述的嵌合多肽的表达。
在本发明的又一方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞包括：携带前述的第一核酸分子或前述的第一表达载体；或，表达前述的嵌合多肽。本发明的重组细胞可有效地表达前述的嵌合多肽。
在本发明的又一方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括前述的嵌合多肽、前述的第一核酸分子、前述的第一表达载体或前述的重组细胞。由前可知，表达上述嵌合多肽的细胞可识别第一分子，其在结合第一分子后可以实现多种不同的信号输出类型，如特定基因的表达激活与抑制等，或者采用表达嵌合多肽的免疫细胞在接触第一分子后，可分泌具有治疗作用的因子(如单链抗体、细胞因子等)，用于抗肿瘤；并且，第一核酸分子和第一表达载体可在细胞(例如免疫细胞)表达嵌合多肽，前述的重组细胞上可表达嵌合多肽。因此，采用含有前述的嵌合多肽、第一核酸分子、第一表达载体或重组细胞的药物组合物可靶向第一分子，用于预防和/或治疗相关疾病，例如用于治疗癌症等。
在本发明的又一方面，本发明提出了一种前述的嵌合多肽、前述的第一核酸分子、前述的第一表达载体、前述的重组细胞或前述的药物组合物在制备药物中用途，所述药物用于预防和/或治疗疾病。由前可知，表达上述嵌合多肽的细胞可识别第一分子，其在结合第一分子后可以实现多种不同的信号输出类型，如特定基因的表达激活与抑制等，或者采用表达嵌合多肽的免疫细胞在接触第一分子后，可分泌具有治疗作用的因子(如单链抗体、细胞因子等)，用于抗肿瘤；并且，第一核酸分子和第一表达载体可在细胞(例如免疫细胞)表达嵌合多肽，前述的重组细胞上可表达嵌合多肽，以及药物组合物包含前述的嵌合多肽、第一核酸分子、第一表达载体或重组细胞。由此，含有嵌合多肽、第一核酸分子、第一表达载体、重组细胞或药物组合物的药物可靶向第一分子，用于预防和/或治疗相关疾病，例如用于治疗癌症等。
在本发明的又一方面，本发明提出了一种前述的嵌合多肽、前述的第一核酸分子、前述的第一表达载体、前述的重组细胞或前述的药物组合物，用于预防和/或治疗疾病。由前可知，表达上述嵌合多肽的细胞可识别第一分子，其在结合第一分子后可以实现多种不同的信号输出类型，如特定基因的表达激活与抑制等，或者采用表达嵌合多肽的免疫细胞在接触第一分子后，可分泌具有治疗作用的因子(如单链抗体、细胞因子等)，用于抗肿瘤；并且，第一核酸分子和第一表达载体可在细胞(例如免疫细胞)表达嵌合多肽，前述的重组细胞上可表达嵌合多肽，以及药物组合物包含前述的嵌合多肽、第一核酸分子、第一表达载体或重组细胞。由此，含有嵌合多肽、第一核酸分子、第一表达载体、重组细胞或药物组合物的药物可靶向第一分子，用于预防和/或治疗相关疾病，例如癌症等。
在本发明的又一方面，本发明提出了一种治疗或预防疾病的方法。根据本发明的实施例。所述方法包括：向受试者施用药学上可接受量的前述的重组细胞或前述的药物组合物。由前可知，表达上述嵌合多肽的细胞可识别第一分子，其在结合第一分子后可以实现多种不同的信号输出类型，如特定基因的表达激活与抑制等，或者采用表达嵌合多肽的免疫细胞在接触第一分子后，可分泌具有治疗作用的因子(如单链抗体、细胞因子等)，用于抗肿瘤；并且，第一核酸分子和第一表达载体可在细胞(例如免疫细胞)表达嵌合多肽，前述的重组细胞上可表达嵌合多肽，以及药物组合物包含前述的嵌合多肽、第一核酸分子、第一表达载体或重组细胞。由此，含有嵌合多肽的重组细胞或药物组合物的药物可靶向第一分子，用于预防和/或治疗相关疾病，例如癌症等。
在本发明的又一方面，本发明提出了一种激活免疫细胞的方法。根据本发明的实施例，所述方法包括：将所述免疫细胞与第一分子进行第一接触，其中，所述免疫细胞表达前述的嵌合多肽。由此，表达上述嵌合多肽的免疫细胞可识别第一分子，其在结合第一分子后可以实现激活免疫细胞。
在本发明的又一方面，本发明提出了一种追踪第一细胞-第二细胞接触的方法。根据本发明的实施例，所述方法包括：将所述第一细胞和所述第二细胞进行第二接触，其中，所述第一细胞表达前述的嵌合多肽和报告基因蛋白，所述嵌合多肽的胞内区为转录激活蛋白，编码所述报告基因蛋白的第三核酸分子的5’端与诱导表达核酸序列与诱导表达核酸序列相连，所述诱导表达核酸序列用于结合所述转录激活蛋白，所述第二细胞表达所述第一分子；基于所述第一细胞中所述报告基因蛋白的检测结果，确定第一细胞和第二细胞的接触情况。由此，采用上述方法可基于报告基因蛋白的检测结果，确定第一细胞和第二细胞之间是否接触。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1为本发明实施例1中SynNotch合成受体的结构示意图；
图2为本发明实施例2中SynNotch合成受体细胞识别特定胞外信号的示意图；
图3为本发明实施例2中膜锚定的GFP的结构示意图；
图4为本发明实施例2中含有鼠Notch跨膜结构域的SynNotch合成受体与含有热带爪蟾Notch跨膜结构域的SynNotch合成受体的泄露效率和有效激活效率；
图5为本发明实施例3中膜锚定的CD19的结构示意图；
图6为本发明实施例3中靶癌细胞对SynNotch-CAR-Jurkat T细胞的激活结果；
图7为本发明实施例4中SynNotch-CAR-NK 92细胞对靶癌细胞的杀伤效率。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个、两个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
在本文中，术语“包含”或“包括”为开放式表达，即包括本发明所指明的内容，但并不排除其他方面的内容。
在本文中，术语“任选地”、“任选的”或“任选”通常是指随后所述的事件或状况可以但未必发生，并且该描述包括其中发生该事件或状况的情况，以及其中未发生该事件或状况的情况。
在本文中，术语“同一性”、“同源性”或“相似性”均用于描述相对于参考序列的氨基酸序列或核酸序列时，采用通过常规的方法进行确定两个氨基酸序列或核酸序列之间的相同氨基酸或核苷酸的百分比，例如参见，Ausubel等，编著(1995)，Current Protocols in Molecular Biology，第19章(Greene Publishing and Wiley-Interscience，New York)；和ALIGN程序(Dayhoff(1978)，Atlas of Protein Sequence and Structure 5：Suppl.3(National Biomedical Research Foundation，Washington，D.C.)。关于比对序列和测定序列同一性有很多算法，包括，Needleman等(1970)J.Mol.Biol.48：443的同源性比对算法；Smith等(1981)Adv.Appl.Math.2：482的局部同源性算法；Pearson等(1988)Proc.Natl.Acad.Sci.85：2444的相似性搜索方法；Smith-Waterman算法(Meth.Mol.Biol.70：173-187(1997)；和BLASTP，BLASTN，和BLASTX算法(参见Altschul等(1990)J.Mol.Biol.215：403-410)。利用这些算法的计算机程序也是可获得的，并且包括但不限于：ALIGN或Megalign(DNASTAR)软件，或者WU-BLAST-2(Altschul等，Meth.Enzym.，266：460-480(1996))；或者GAP，BESTFIT，BLAST Altschul等，上文，FASTA，和TFASTA，在Genetics Computing Group(GCG)包，8版，Madison，Wisconsin，USA中可获得；和Intelligenetics，Mountain View，California提供的PC/Gene程序中的CLUSTAL。
在本文中，术语“至少80％同一性”指与各参考序列至少为80％，可为80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、99.9％的同一性。
在本文中，术语“表达载体”通常是指能够插入在合适的宿主中自我复制的核酸分子，其将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述表达载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述表达载体还包括具有多种上述功能的载体。所述表达载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述表达载 体的合适的宿主细胞，所述表达载体可以产生期望的表达产物。
在本文中，术语“重组细胞”通常是指采用基因工程技术或细胞融合技术对宿主细胞的遗传物质进行修饰改造或重组，获得具有稳定遗传的独特性状的细胞。其中，术语“宿主细胞”是指可导入重组表达载体的原核细胞或真核细胞。本文所用术语“转化的”或“转染的”是指通过本领域已知的各种技术将核酸(例如载体)引入细胞。合适的宿主细胞可以用本发明的DNA序列转化或转染，并且可以用于靶蛋白的表达和/或分泌。
在本文中，术语“药物组合物”通常是指单位剂量形式，并且可以通过制药领域中熟知的方法的任何一种进行制备。所有的方法包括使活性成分与构成一种或多种附属成分的载体相结合的步骤。通常，通过均匀并充分地使活性嵌合多肽或重组细胞与液体载体、细碎固体载体或这两者相结合，制备组合物。
在本文中，术语“药学上可接受的辅料”均可包括任何溶剂、固体赋形剂、稀释剂或其他液体赋形剂等等，适合于特有的目标剂型。除了任何常规的辅料与本发明的嵌合多肽或重组细胞不相容的范围，例如所产生的任何不良的生物效应或与药学上可接受的组合物的任何其他组分以有害的方式产生的相互作用，它们的用途也是本发明所考虑的范围。
在本文中，术语“给药”指将预定量的物质通过某种适合的方式引入病人。本发明的重组细胞或药物组合物可以通过任何常见的途径被给药，只要它可以到达预期的组织。给药的各种方式是可以预期的，包括腹膜、静脉注射、肌肉注射、皮下注射等等，但是本发明不限于这些已举例的给药方式。优选地，本发明的组合物采用静脉注射或皮下注射方式被给药。
在本文中，术语“治疗”是指用于获得期望的药理学和/或生理学效果。所述效果就完全或部分预防疾病或其症状而言可以是预防性的，和/或就部分或完全治愈疾病和/或疾病导致的不良作用而言可以是治疗性的。本文使用的“治疗”涵盖哺乳动物、特别是人的疾病，包括：(a)在容易患病但是尚未确诊得病的个体中预防疾病或病症发生；(b)抑制疾病，例如阻滞疾病发展；或(c)缓解疾病，例如减轻与疾病相关的症状。本文使用的“治疗”涵盖将嵌合多肽、重组细胞、药物组合物或药物给予个体以治疗、治愈、缓解、改善、减轻或抑制个体的疾病的任何用药，包括但不限于将含本文所述嵌合多肽、重组细胞或药物组合物的药物给予有需要的个体。
本发明提出了一种分离的多肽在制备SynNotch合成受体中的用途、嵌合多肽、第一核酸分子、第一表达载体、重组细胞、药物组合物及其用途、激活免疫细胞的方法、追踪第一细胞-第二细胞接触的方法，下面将分别对其进行详细描述。
分离的多肽在制备SynNotch合成受体中的用途
在本发明的一个方面，本发明提出了一种分离的多肽在制备SynNotch合成受体中的用途，所述分离的多肽与来源于热带爪蟾(Xenopus tropicalis)的Notch受体蛋白的跨膜区具有至少80％的同一性。发明人发现，选择热带爪蟾的Notch受体蛋白的跨膜区制备的SynNotch合成受体，可以提高其对下游基因转录激活的能力和效率，以及降低SynNotch合成受体的本底泄露。
根据本发明的实施例，所述分离的多肽与来源于热带爪蟾(Xenopus tropicalis)的Notch受体蛋白的跨膜区具有100％的同一性。
根据本发明的实施例，所述来源于热带爪蟾(Xenopus tropicalis)的Notch受体蛋白的跨膜区具有如SEQ ID NO:1所示的氨基酸序列。
嵌合多肽
在本发明的另一方面，本发明提出了一种嵌合多肽。根据本发明的实施例，所述嵌合多肽包括：胞外区，所述胞外区具有结合第一分子活性；跨膜区，所述跨膜区的N端与所述胞外区的C端相连；胞内区，所述胞内区的N端与所述跨膜区的C端相连；其中，所述跨膜区包括分离的多肽，所述分离的多肽与来源于热带爪蟾的Notch受体蛋白的跨膜区具有至少80％的同一性。
发明人发现，选择热带爪蟾的Notch受体蛋白的跨膜区作为嵌合多肽(即为SynNotch合成受体)的跨膜区，可以提高嵌合多肽对下游基因转录激活的能力和效率，以及降低嵌合多肽的本底泄露。并且，使表达该嵌合多肽的细胞可识别 第一分子，其在结合第一分子后可以实现多种不同的信号输出类型，如特定基因的表达激活与抑制等，以及用于制备表达嵌合多肽的免疫细胞，该免疫细胞在接触第一分子后，可分泌具有治疗作用的因子(如单链抗体、细胞因子等)，用于抗肿瘤。
根据本发明的实施例，所述分离的多肽与来源于热带爪蟾(Xenopus tropicalis)的Notch受体蛋白的跨膜区具有100％的同一性。
根据本发明的实施例，所述来源于热带爪蟾(Xenopus tropicalis)的Notch受体蛋白的跨膜区具有如SEQ ID NO:1所示的氨基酸序列。
根据本发明的实施例，所述跨膜区进一步包括表皮生长因子样重复序列(EGF repeat)和/或RAM序列。由此，可进一步降低SynNotch合成受体的本底泄露激活。
根据本发明的实施例，所述表皮生长因子样重复序列如SEQ ID NO:15所示的氨基酸序列。
根据本发明的实施例，所述RAM序列如SEQ ID NO:16所示的氨基酸序列。
根据本发明的实施例，所述表皮生长因子样重复序列的C端和所述分离的多肽的N端相连；和/或，所述分离的多肽的C端和所述RAM序列的N端相连。
根据本发明的实施例，所述第一分子包括肿瘤抗原、病毒、细菌、内毒素、抗体、细胞受体和细胞受体的配体中的至少之一。
在本文中，术语“肿瘤抗原”通常是指在肿瘤发生、发展过程中新出现或过度表达的抗原物质。根据肿瘤抗原特异性的分类法分为肿瘤特异性抗原和肿瘤相关抗原，其中，肿瘤特异性抗原(tumor specific antigen,TSA)是肿瘤细胞特有的或只存在于某种肿瘤细胞而不存在于正常细胞的新抗原，肿瘤相关抗原(tumor-associated antigen,TAA)是指非肿瘤细胞所特有的、正常细胞和其他组织上也存在的抗原，只是其含量在细胞癌变时明显增高。其中，包括但不限于PD-L1、PD-1、TGF-β、CEA、GD2和GD3等。
在本文中，术语“细胞受体”或“受体”应做广义理解，可以是指位于细胞膜上、可识别细胞外的各种信号分子(配体)并与之结合的分子，包括但不限于生长因子受体(如VEGF受体)、(NKG2D多肽(MICA、MICB和ULB6的受体)、细胞因子受体(如IL-13受体、IL-2受体等)、表皮生长因子(EGF)受体、Her2、CD27、天然细胞毒性受体(NCR)(如NKP30(NCR3/CD337)多肽(HLA-B相关转录物3(BAT3)和B7-H6的受体)等)、T细胞抗原受体、二氢叶酸受体、嵌合细胞因子受体、Fc受体、细胞外基质受体(例如整联蛋白)、细胞粘附受体(例如钙粘蛋白)、免疫调节受体(包括正辅助受体(例如CD28)和负(免疫抑制)辅助受体(例如PD1))以及免疫调节分子(例如TGFβ)的受体等。
在本文中，术语“细胞受体的配体”应做广义理解，可以是指能与细胞膜受体结合、相互作用并产生特定生物学效应的化学物质，例如多肽、核酸、糖蛋白、小分子、碳水化合物、脂质、糖脂、脂蛋白、脂多糖等，包括但不限于细胞因子(例如，IL-13等)、生长因子(例如，heregulin、血管内皮生长因子(VEGF)等)、肽激素、整联蛋白结合肽(例如包括序列Arg-Gly-Asp的肽)、N-聚糖等。
示例性地，配体为VEGF、受体为VEGF受体；或者配体为heregulin、受体为Her2。
在本文中，术语“细胞因子”应做广义理解，可以是指一类能在细胞间传递信息、具有免疫调节和效应功能的蛋白质或小分子多肽，例如IL-10。“细胞因子受体”应做广义理解，可以是指细胞表面上可与细胞因子结合的受体，例如Her2和IL-10R等。
在本发明的一个优选实施例中，所述肿瘤抗原为肿瘤特异性抗原。
根据本发明的实施例，所述第一分子包括GFP、eGFP、CD19、ALPPL2、BCMA、SIRPα、CD1a、CD1b、CD1c、CD1d、CD1e、CD2、CD3d、CD3e、CD3g、CD4、CD5、CD7、CD8a、CD8b、CD20、CD21、CD22、CD23、CD25、CD27、CD28、CD30、CD33、CD34、CD38、CD40、CD44、CD44v6、CD45、CD48、CD51、CD52、CD56、CD59、CD66、CD70、CD71、CD72、CD73、CD74、CD79A、CD79B、CD80、CD86、CD94、CD95、CD133、CD134、CD140、CD152、CD154、CD158、CD178、CD181、CD182、CD183、CD200、CD210、CD221、CD246、CD252、CD253、CD261、CD262、CD273、CD274、CD276、CD279、CD295、CD339、CD340、EGFR、HER2、FGFR2、AFP、CA125、MSLN、GPC3、CEA、CLDN18.2、EpCAM、PSCA、GD2、IL-13、IL-13RA2、ROR1、MUC 1、PSMA、MAGEA1、4-1BB、5T4、BAFF、CA242、CA-IX、MET、CCR4、CNTO888、FAP、MORAb-009、VEGF-A、VEGFR-1和VEGFR-2中的至少之一。
根据本发明的实施例，所述胞外区包括结合所述第一分子的第一结合蛋白或其片段；
根据本发明的实施例，所述第一结合蛋白或其片段包括抗体或其功能性片段、受体、受体的配体和细胞黏附分子中的至少之一。
在本文中，“细胞黏附分子”或“CAM”可以是指结合细胞外基质(ECM)的组分或结合细胞表面分子的多肽。例如，细胞黏附分子可为CAM的细胞外区域，其中，CAM可以为不依赖钙的粘附分子，例如CAM是免疫球蛋白超级族CAM，CAM还可以为钙依赖性粘附分子，例如CAM是整联蛋白、钙粘蛋白或选择素；细胞黏附分子可为整联蛋白，例如为钙粘蛋白，例如E-钙粘蛋白、P-钙粘蛋白、N-钙粘蛋白、R-钙粘蛋白、M-钙粘蛋白等；细胞黏附分子还可为选择素，例如E-选择素、L-选择素或P-选择素。
在本文中，术语“抗体”在最广义上使用，其可以包括全长单克隆抗体、多特异性抗体、以及嵌合抗体，具体结构不受限制，只要它们展示所需的生物学活性。其通常包括分子量较轻的轻链和分子量较重的重链，重链(H链)和轻链(L链)由二硫键连接形成的抗体分子。其中，肽链的氨基端(N端)氨基酸序列变化很大，称为可变区(V区)；羧基端(C端)相对稳定，变化很小，称为恒定区(C区)。L链和H链的V区分别称为VL和VH。
在本文中，术语“全长抗体”、“全长单抗”或“全长单克隆抗体”均是由至少两条相同的轻链和至少两条相同的重链通过链间二硫键连接而成，如免疫球蛋白G(IgG)、免疫球蛋白A(IgA)、免疫球蛋白M(IgM)、免疫球蛋白D(IgD)或免疫球蛋白E(IgE)。
在本文中，术语“功能性片段”是包含抗体的一部分或全部的片段，其缺乏至少一些存在于全长链中的氨基酸但仍能够特异性结合抗原的性能活性，例如，该片段可包含抗体CDR的一部分或全部。此类片段具生物活性，因为其结合至抗原，且可与其他抗原结合分子(包括完整抗体)竞争结合至给定表位。此类片段选自Fab、Fv、scFv或单域抗体。此类片段可通过重组核酸技术产生，或可通过抗原结合分子(包括完整抗体)的酶裂解或化学裂解产生。
根据本发明的实施例，所述第一结合蛋白或其片段为结合所述第一分子的单抗或多抗。
在本文中，术语“多抗”和“多特异性抗体”同义，均是指可识别多种抗原表位的抗体，例如可识别两种抗原表位的抗体(双特异性抗体，简称双抗)、三种抗原表位的抗体或者四种抗原表位的抗体，其为广义理解，具体结构不受限制，可识别多种抗原表位即可。在本发明中，多种抗原表位的其中至少之一来源于第一分子。
根据本发明的实施例，所述单抗包括Fab抗体、F(ab')2片段、Fv抗体、单链抗体、单域抗体以及最小识别单位的至少之一。
在本文中，术语“单域抗体”、“纳米抗体”和“VHH抗体”可互换使用，其最初被描述为“重链抗体”(即“缺乏轻链的抗体”)的抗原结合免疫球蛋白(可变)域(Hamers-Casterman C,AtarhouchT,Muyldermans S,Robinson G,Hamers C,Songa EB,Bendahman N,Hamers R.:“Naturallyoccurring antibodies devoid of light chains”；Nature 363,446-448(1993))，包含重链可变区(VH)和常规的CH2与CH3区，其通过重链可变区与抗原蛋白(例如D-二聚体)特异性结合。
在本文中，术语“Fab抗体”或“Fab片段”通常是指仅含Fab分子的抗体或片段，其由重链的VH和CH1以及完整的轻链构成，轻链和重链之间通过一个二硫键连接。
在本文中，术语“F(ab’)2抗体”或“F(ab’)2片段”具有通过二硫键连接在一起的两个抗原结合F(ab’)部分。
在本文中，术语“Fv抗体”或“Fv片段”通常是指仅由轻链可变区(VL)和重链可变区(VH)通过非共价键连接而成的抗体或片段，是抗体分了保留完整抗原结合部位的最小功能片段。
在本文中，术语“单链抗体”、“scFv片段”是由抗体重链可变区和轻链可变区通过短肽连接而成的抗体或片段。
在本文中，术语“最小识别单位”和“MRU”均是指仅由一个CDR组成的抗体或片段，其分子量十分小仅占完全抗体的1％左右。
根据本发明的实施例，所述胞内区包括转录激活蛋白、转录阻遏蛋白、转录因子、位点特异性核酸酶、重组酶、活化性免疫受体胞内结构域和抑制性免疫受体胞内结构域中的至少之一。
根据本发明的实施例，所述胞内区包括GaL4 VP64、GaL4 VP16、tetR VP64、ZFHD1 VP64、Gal4 KRAB、HAP1 VP16、LexA-VP64、Cas9和Cas13中的至少之一。
根据本发明的实施例，所述Gal4 VP64具有如SEQ ID NO:2所示的氨基酸序列。
根据本发明的实施例，所述跨膜区和胞内区具有如SEQ ID NO:3所示的氨基酸序列。
根据本发明的实施例，所述嵌合多肽具有如SEQ ID NO:4所示的氨基酸序列。
核酸分子、载体、重组细胞和药物组合物
在本发明的又一方面，本发明提出了一种第一核酸分子。根据本发明的实施例，所述第一核酸分子编码权利要求前述的嵌合多肽。根据本发明实施例的第一核酸分子可编码获得上述的嵌合多肽。
根据本发明的实施例，所述核酸分子为DNA。
需要说明的是，对于本文中所提及的第一核酸分子，本领域技术人员应当理解，实际包括互补双链的任意一条，或者两条。为了方便，在本文中，虽然多数情况下只给出了一条链，但实际上也公开了与之互补的另一条链。另外，本发明中的分子序列包括DNA形式或RNA形式，公开其中一种，意味着另一种也被公开。
在本发明的又一方面，本发明提出了一种第一表达载体。根据本发明的实施例，所述第一表达载体携带前述的第一核酸分子。在将上述第一核酸分子连接到载体上时，可以将第一核酸分子与载体上的控制元件直接或者间接相连，只要这些控制元件能够控制第一核酸分子的翻译和表达等即可。当然这些控制元件可以直接来自于载体本身，也可以是外源性的，即并非来自于载体本身。当然，第一核酸分子与控制元件进行可操作地连接即可。
本文中“可操作地连接”是指将外源基因连接到载体上，使得载体内的控制元件，例如转录控制序列和翻译控制序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。常用的载体例如可以为质粒、噬菌体等等。根据本发明的一些具体实施例的载体导入合适的接收细胞(又称为受体细胞或宿主细胞)后，可在调控系统的介导下，有效实现前述的嵌合多肽的表达。
根据本发明的实施例，所述第一表达载体为真核表达载体、原核表达载体、病毒或噬菌体。
根据本发明的实施例，所述第一表达载体为质粒表达载体。
在本发明的又一方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞包括：携带前述的第一核酸分子或前述的第一表达载体；或，表达前述的嵌合多肽。利用该重组细胞在适合条件下，能够在重组细胞内有效地表达前述的嵌合多肽。
需要说明的是，本发明中所述的“适合条件”，是指适合前述的嵌合多肽表达的条件。本领域技术人员容易理解的是，适合前述的嵌合多肽表达的条件包括但不限于合适的转化或转染方式、合适的转化或转染条件、健康的细胞状态、合适的细胞密度、适宜的细胞培养环境、适宜的细胞培养时间。“适合条件”不受特别限制，本领域技术人员可根据实验室的具体环境，优化最适的嵌合多肽表达的条件。
根据本发明的实施例，所述重组细胞是通过将前述的第一表达载体引入至宿主细胞中而获得的。
根据本发明的实施例，所述宿主细胞包括免疫细胞、神经元、祖细胞或前体细胞、上皮细胞、内皮细胞和干细胞中 的至少之一。
根据本发明的实施例，所述宿主细胞包括T细胞、B细胞、单核细胞、NK细胞、树突细胞、巨噬细胞、调节T细胞、辅助T细胞、细胞毒性T细胞、NKT细胞和γδT细胞中的至少之一。
根据本发明的实施例，所述重组细胞进一步包括：编码嵌合抗原受体、NK细胞受体或T细胞受体的第二核酸分子或携带所述第二核酸分子的第二表达载体；或表达所述嵌合抗原受体、T细胞受体或NK细胞受体。发明人经过试验发现，将本发明的嵌合多肽与嵌合抗原受体、NK细胞受体或T细胞受体进行联用，共同改造免疫细胞(如Jurkat细胞和NK-92细胞)，发现嵌合多肽+CAR/TCR联用的免疫细胞可识别相应的配体，并高效激活免疫细胞(如Jurkat细胞和NK-92细胞)，以行使杀伤目标(如肿瘤细胞等)的功能。
在本文中，术语“嵌合抗原受体(CAR)”是一种融合蛋白，其包含能够结合抗原的胞外结构域，与胞外结构域衍生自不同多肽的跨膜结构域，以及至少一个胞内结构域。“嵌合抗原受体(CAR)”也称为“嵌合受体”、“T-体”或“嵌合免疫受体(CIR)”。所述的“能够结合抗原的胞外结构域”是指能够结合某一抗原的任何寡肽或多肽。“胞内结构域”是指已知的作为传递信号以激活或抑制细胞内生物过程的结构域起作用的任何寡肽或多肽。
本文中，术语“T细胞受体(简称TCR)”是一种可以在T细胞表面上找到的分子，其负责识别可与MHC分子结合的抗原。天然存在的TCR异二聚体由约95％的T细胞中的alpha(α)和beta(β)链组成，而约5％的T细胞具有由gamma(γ)和delta(δ)链组成的TCR。在抗原加工过程中，抗原在细胞内被降解，然后被主要组织相容性复合物(MHC)分子携带到细胞表面，T细胞能够识别抗原呈递细胞表面上的此抗原肽-MHC复合物，TCR与抗原肽-MHC复合物的结合导致T淋巴细胞活化，在T淋巴细胞上通过由相关的酶、共受体和专门的辅助分子介导的一系列生化反应来表达TCR。其中，MHC分子可以是I类或II类MHC分子。该复合物可以在抗原呈递细胞，例如：树突状细胞、B细胞或任何其他细胞(如K562细胞)。人白细胞抗原系统(HLA)是编码人类主要组织相容性复合物(MHC)的基因复合物的名称，并且包括HLA I类抗原(A、B和C)和HLA II类抗原(DP、DQ和DR)。HLA等位基因A、B和C呈递主要源自细胞内蛋白质，例如在细胞内表达的蛋白质的肽。
本文中，术语“NK细胞受体”是一种可以在NK细胞表面上找到的分子，且根据其所介导的功能不同又可分为抑制性受体和激活性受体。其中，抑制性受体用于识别正常细胞表面表达的特定分子后，转导杀伤抑制信号，抑制自身的杀伤功能；抑制性受体种类较多，包括但不限于CD161、CLRG1、PD1、TIM3、LAG 3、CD96和TIGIT。激活性受体用于识别靶细胞表面对应的配体后，向细胞内转导活化信号，发挥杀伤作用。激活受体包括但不限于NKp30、NKp44、NKp46和CD16。
根据本发明的实施例，所述胞内区包括转录激活蛋白，编码所述嵌合抗原受体、NK细胞受体或T细胞受体的第二核酸分子的5’端与诱导表达核酸序列相连，所述诱导表达核酸序列用于结合所述转录激活蛋白。
示例性地，所述胞内区为GaL4 VP64，所述诱导表达核酸序列为UAS-minimal-CMV序列。
所述UAS-minimal-CMV序列具有如SEQ ID NO:14所示的核酸序列。
在本发明的一个可选实施例中，所述嵌合抗原受体、NK细胞受体或T细胞受体具有结合第二分子活性。例如，嵌合抗原受体主要通过其胞外区结合第二分子。
根据本发明的实施例，所述第二分子包括肿瘤抗原、病毒、细菌、内毒素、抗体、细胞受体和细胞受体的配体中的至少之一。
在本发明的一个可选实施例中，所述第二分子包括GFP、eGFP、CD19、ALPPL2、BCMA、SIRPα、CD1a、CD1b、CD1c、CD1d、CD1e、CD2、CD3d、CD3e、CD3g、CD4、CD5、CD7、CD8a、CD8b、CD20、CD21、CD22、CD23、CD25、CD27、CD28、CD30、CD33、CD34、CD38、CD40、CD44、CD44v6、CD45、CD48、CD51、CD52、CD56、CD59、CD66、CD70、CD71、CD72、CD73、CD74、CD79A、CD79B、CD80、CD86、CD94、CD95、CD133、CD134、CD140、CD152、CD154、CD158、CD178、CD181、CD182、CD183、CD200、CD210、CD221、CD246、CD252、CD253、CD261、CD262、CD273、CD274、CD276、CD279、CD295、CD339、CD340、EGFR、HER2、FGFR2、AFP、CA125、MSLN、GPC3、CEA、CLDN18.2、EpCAM、PSCA、GD2、IL-13、IL-13RA2、ROR1、MUC 1、PSMA、MAGEA1、4-1BB、5T4、BAFF、CA242、CA-IX、MET、CCR4、CNTO888、FAP、MORAb-009、VEGF-A、VEGFR-1和VEGFR-2 中的至少之一。
在本发明的又一方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括前述的嵌合多肽、前述的第一核酸分子、前述的第一表达载体或前述的重组细胞。由前可知，表达上述嵌合多肽的细胞可识别第一分子，其在结合第一分子后可以实现多种不同的信号输出类型，如特定基因的表达激活与抑制等，或者采用表达嵌合多肽的免疫细胞在接触第一分子后，可分泌具有治疗作用的因子(如单链抗体、细胞因子等)，用于抗肿瘤；并且，第一核酸分子和第一表达载体可在细胞(例如免疫细胞)表达嵌合多肽，前述的重组细胞上可表达嵌合多肽。因此，采用含有前述的嵌合多肽、第一核酸分子、第一表达载体或重组细胞的药物组合物可靶向第一分子，用于预防和/或治疗相关疾病，例如癌症等。
根据本发明的实施例，进一步包括药学上可接受的辅料。
用途
在本发明的又一方面，本发明提出了一种前述的嵌合多肽、前述的第一核酸分子、前述的第一表达载体、前述的重组细胞或前述的药物组合物在制备药物中用途，所述药物用于预防和/或治疗疾病。由前可知，表达上述嵌合多肽的细胞可识别第一分子，其在结合第一分子后可以实现多种不同的信号输出类型，如特定基因的表达激活与抑制等，或者采用表达嵌合多肽的免疫细胞在接触第一分子后，可分泌具有治疗作用的因子(如单链抗体、细胞因子等)，用于抗肿瘤；并且，第一核酸分子和第一表达载体可在细胞(例如免疫细胞)表达嵌合多肽，前述的重组细胞上可表达嵌合多肽，以及药物组合物包含前述的嵌合多肽、第一核酸分子、第一表达载体或重组细胞。由此，含有嵌合多肽、第一核酸分子、第一表达载体、重组细胞或药物组合物的药物可靶向第一分子，用于预防和/或治疗相关疾病，例如癌症等。
在本发明的又一方面，本发明提出了一种前述的嵌合多肽、前述的第一核酸分子、前述的第一表达载体、前述的重组细胞或前述的药物组合物，用于预防和/或治疗疾病。由前可知，表达上述嵌合多肽的细胞可识别第一分子，其在结合第一分子后可以实现多种不同的信号输出类型，如特定基因的表达激活与抑制等，或者采用表达嵌合多肽的免疫细胞在接触第一分子后，可分泌具有治疗作用的因子(如单链抗体、细胞因子等)，用于抗肿瘤；并且，第一核酸分子和第一表达载体可在细胞(例如免疫细胞)表达嵌合多肽，前述的重组细胞上可表达嵌合多肽，以及药物组合物包含前述的嵌合多肽、第一核酸分子、第一表达载体或重组细胞。由此，含有嵌合多肽、第一核酸分子、第一表达载体、重组细胞或药物组合物的药物可靶向第一分子，用于预防和/或治疗相关疾病，例如癌症等。
根据本发明的实施例，上述两个方面的用途还可以进一步包括如下技术特征的至少之一：
根据本发明的实施例，所述疾病包括癌症或肿瘤、免疫相关疾病。
根据本发明的实施例，所述疾病包括癌症或肿瘤、自身免疫性疾病、炎症及由细胞衰老所引发的相关疾病。
在本文中，术语“癌症”或“肿瘤”均可以是任何不受调控的细胞生长。示例性地，可以是非小细胞肺癌、乳头状甲状腺癌、多形性成胶质细胞瘤、结肠癌、直肠癌、肺癌、头颈癌、肾癌、膀胱癌、乳腺癌、卵巢癌、肝癌、胆管癌或肉瘤、急性骨髓性白血病、大细胞神经内分泌癌、成神经细胞瘤、前列腺癌、成神经细胞瘤、胰腺癌、黑色素瘤、头颈鳞状细胞癌、宫颈癌、皮肤癌、神经胶质瘤、食道癌、口腔鳞状细胞癌或胃癌等等。
方法
在本发明的又一方面，本发明提出了一种激活免疫细胞的方法。根据本发明的实施例，所述方法包括：将所述免疫细胞与第一分子进行第一接触，其中，所述免疫细胞表达前述的嵌合多肽。由此，表达上述嵌合多肽的免疫细胞可识别第一分子，其在结合第一分子后可以实现激活免疫细胞，尤其是适用于科学研究，通过对免疫细胞进行体外细胞培养，以便获得被激活的免疫细胞。
根据本发明的实施例，所述嵌合多肽的胞内区包括转录激活蛋白，所述免疫细胞表达嵌合抗原受体、NK细胞受体或T细胞受体，所述嵌合抗原受体、NK细胞受体或T细胞受体包括结合预定抗原的抗体或其功能性片段，编码所述嵌合抗原受体、NK细胞受体或T细胞受体的第二核酸分子的5’端与诱导表达核酸序列相连，所述诱导表达核酸序列用于结合所述转录激活蛋白；所述方法进一步包括：所述第一接触之后，所述免疫细胞释放所述转录激活蛋白，并表达所述嵌合抗原受体、NK细胞受体或T细胞受体；将所述免疫细胞的嵌合抗原受体、NK细胞受体或T细胞受体与预定抗原结合，以便激活所述免疫细胞。
根据本发明的实施例，所述诱导表达核酸序列具有如SEQ ID NO:14所示的核酸序列。
根据本发明的实施例，所述第一分子为肿瘤抗原。
在本发明的一个可选实施例中，所述第一分子包括GFP、eGFP、CD19、ALPPL2、BCMA、SIRPα、CD1a、CD1b、CD1c、CD1d、CD1e、CD2、CD3d、CD3e、CD3g、CD4、CD5、CD7、CD8a、CD8b、CD20、CD21、CD22、CD23、CD25、CD27、CD28、CD30、CD33、CD34、CD38、CD40、CD44、CD44v6、CD45、CD48、CD51、CD52、CD56、CD59、CD66、CD70、CD71、CD72、CD73、CD74、CD79A、CD79B、CD80、CD86、CD94、CD95、CD133、CD134、CD140、CD152、CD154、CD158、CD178、CD181、CD182、CD183、CD200、CD210、CD221、CD246、CD252、CD253、CD261、CD262、CD273、CD274、CD276、CD279、CD295、CD339、CD340、EGFR、HER2、FGFR2、AFP、CA125、MSLN、GPC3、CEA、CLDN18.2、EpCAM、PSCA、GD2、IL-13、IL-13RA2、ROR1、MUC 1、PSMA、MAGEA1、4-1BB、5T4、BAFF、CA242、CA-IX、MET、CCR4、CNTO888、FAP、MORAb-009、VEGF-A、VEGFR-1和VEGFR-2中的至少之一。
根据本发明的实施例，所述免疫细胞包括T细胞、B细胞、单核细胞、NK细胞、树突细胞、巨噬细胞、调节T细胞、辅助T细胞、细胞毒性T细胞、NKT细胞和γδT细胞中的至少之一。
在本发明的又一方面，本发明提出了一种追踪第一细胞-第二细胞接触的方法。根据本发明的实施例，所述方法包括：将所述第一细胞和所述第二细胞进行第二接触，其中，所述第一细胞表达前述的嵌合多肽和报告基因蛋白，所述嵌合多肽的胞内区为转录激活蛋白，编码所述报告基因蛋白的第三核酸分子的5’端与诱导表达核酸序列相连，所述诱导表达核酸序列用于结合所述转录激活蛋白，所述第二细胞表达所述第一分子；基于所述第一细胞中所述报告基因蛋白的检测结果，确定第一细胞和第二细胞的接触情况。由此，采用上述方法可基于报告基因蛋白的检测结果，确定第一细胞和第二细胞之间是否接触，尤其是适用于科学研究，可通过该方法用于体外判断两种细胞之间是否进行了接触。
根据本发明的实施例，所述诱导表达核酸序列具有如SEQ ID NO:14所示的核酸序列。
根据本发明的实施例，所述第一分子在第二细胞的表面上、固定在不溶性基材上、存在于细胞外基质中、存在于人工基质中，或是可溶的。
根据本发明的实施例，所述第一分子为肿瘤抗原。
在本发明的一个可选实施例中，所述第一分子包括GFP、eGFP、CD19、ALPPL2、BCMA、SIRPα、CD1a、CD1b、CD1c、CD1d、CD1e、CD2、CD3d、CD3e、CD3g、CD4、CD5、CD7、CD8a、CD8b、CD20、CD21、CD22、CD23、CD25、CD27、CD28、CD30、CD33、CD34、CD38、CD40、CD44、CD44v6、CD45、CD48、CD51、CD52、CD56、CD59、CD66、CD70、CD71、CD72、CD73、CD74、CD79A、CD79B、CD80、CD86、CD94、CD95、CD133、CD134、CD140、CD152、CD154、CD158、CD178、CD181、CD182、CD183、CD200、CD210、CD221、CD246、CD252、CD253、CD261、CD262、CD273、CD274、CD276、CD279、CD295、CD339、CD340、EGFR、HER2、FGFR2、AFP、CA125、MSLN、GPC3、CEA、CLDN18.2、EpCAM、PSCA、GD2、IL-13、IL-13RA2、ROR1、MUC 1、PSMA、MAGEA1、4-1BB、5T4、BAFF、CA242、CA-IX、MET、CCR4、CNTO888、FAP、MORAb-009、VEGF-A、VEGFR-1和VEGFR-2中的至少之一。
根据本发明的实施例，所述第一细胞或第二细胞包括免疫细胞、神经元、祖细胞或前体细胞、上皮细胞、内皮细胞和干细胞中的至少之一。
根据本发明的实施例，所述第一细胞包括T细胞、B细胞、单核细胞、NK细胞、树突细胞、巨噬细胞、调节T细胞、辅助T细胞、细胞毒性T细胞、NKT细胞和γδT细胞中的至少之一。
在本发明的又一方面，本发明提出了一种调控细胞活性的方法。根据本发明的实施例，所述方法包括：将所述细胞与第一分子进行第三接触，其中，所述细胞表达前述的嵌合多肽。
根据本发明的实施例，所述细胞包括免疫细胞、神经元、祖细胞或前体细胞、上皮细胞、内皮细胞和干细胞中的至少之一。
根据本发明的实施例，所述细胞包括T细胞、B细胞、单核细胞、NK细胞、树突细胞、巨噬细胞、调节T细胞、辅助T细胞、细胞毒性T细胞、NKT细胞和γδT细胞中的至少之一。
在本发明的又一方面，本发明提出了一种促进细胞内基因或蛋白表达的方法。根据本发明的实施例，所述方法包括：将所述细胞与第一分子进行第四接触，其中，所述细胞表达前述的嵌合多肽，所述细胞携带所述基因或表达所述蛋白，其中，所述基因包括编码嵌合抗原受体、第二嵌合多肽、翻译调控子、细胞因子、激素、趋化因子、抗体和位于所述胞内区的蛋白中的至少之一的核酸分子，或者，所述蛋白包括嵌合抗原受体、第二嵌合多肽、翻译调控子、细胞因子、激素、趋化因子、抗体和位于所述胞内区的蛋白中的至少之一。
根据本发明的实施例，所述嵌合多肽的胞内区为转录激活蛋白，所述嵌合抗原受体、第二嵌合多肽、翻译调控子、 细胞因子、激素、趋化因子或抗体包含所述转录激活蛋白的第二结合蛋白或其片段。
根据本发明的实施例，所述细胞包括免疫细胞、神经元、祖细胞或前体细胞、上皮细胞、内皮细胞和干细胞中的至少之一。
根据本发明的实施例，所述细胞包括T细胞、B细胞、单核细胞、NK细胞、树突细胞、巨噬细胞、调节T细胞、辅助T细胞、细胞毒性T细胞、NKT细胞和γδT细胞中的至少之一。
在本发明的又一方面，本发明提出了一种治疗或预防疾病的方法。根据本发明的实施例。所述方法包括：向受试者施用药学上可接受量的前述的重组细胞或前述的药物组合物。由前可知，表达上述嵌合多肽的细胞可识别第一分子，其在结合第一分子后可以实现多种不同的信号输出类型，如特定基因的表达激活与抑制等，或者采用表达嵌合多肽的免疫细胞在接触第一分子后，可分泌具有治疗作用的因子(如单链抗体、细胞因子等)，用于抗肿瘤；并且，第一核酸分子和第一表达载体可在细胞(例如免疫细胞)表达嵌合多肽，前述的重组细胞上可表达嵌合多肽，以及药物组合物包含前述的嵌合多肽、第一核酸分子、第一表达载体或重组细胞。由此，含有嵌合多肽的重组细胞或药物组合物的药物可靶向第一分子，用于预防和/或治疗相关疾病，例如癌症等。
本发明所述的重组蛋白或药物组合物的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
本发明的重组蛋白或药物组合物可掺入适用于胃肠外施用(例如静脉内、皮下、腹膜内、肌肉内)的药物中。这些药物可以被制备成各种形式。例如液体、半固体和固体剂型等，包括但不限于液体溶液(例如，注射溶液和输注溶液)或冻干粉。典型的药物为注射溶液或输注溶液形式。前述重组蛋白或药物组合物可通过静脉输注或注射或肌肉内或皮下注射来施用。
根据本发明的实施例，所述方法的给药途径采用皮下注射或静脉注射。
根据本发明的实施例，所述疾病包括癌症或肿瘤、免疫相关疾病。根据本发明的实施例，所述疾病包括癌症或肿瘤、自身免疫性疾病、炎症及由细胞衰老所引发的相关疾病。
根据本发明的实施例，所述肿瘤或癌症包括但不限于非小细胞肺癌、乳头状甲状腺癌、多形性成胶质细胞瘤、结肠癌、直肠癌、肺癌、头颈癌、肾癌、膀胱癌、乳腺癌、卵巢癌、肝癌、胆管癌或肉瘤、急性骨髓性白血病、大细胞神经内分泌癌、成神经细胞瘤、前列腺癌、成神经细胞瘤、胰腺癌、黑色素瘤、头颈鳞状细胞癌、宫颈癌、皮肤癌、神经胶质瘤、食道癌、口腔鳞状细胞癌或胃癌等。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：SynNotch合成受体设计及其表达质粒载体构建
1、SynNotch合成受体(简称SynNotch受体)的结构包括胞外域(又称胞外区)、Notch跨膜结构域(又称Notch受体蛋白的跨膜区，简称跨膜区)和胞内域(又称胞内区)，其中，胞外域可用单链抗体或者纳米抗体等替换，胞内域可用转录激活或转录抑制的结构域等替换。本发明以anti-GFP单链抗体作为胞外域部分，以GAL4-VP64作为胞内域部分，并以能够被蛋白酶识别切割的鼠或热带爪蟾或Notch跨膜结构域作为Notch跨膜结构域，获得改造后的SynNotch受体，本发明设计改造的SynNotch受体的结构如图1所示。图1显示了包含有鼠或热带爪蟾的Notch跨膜结构域(Notch core)的SynNotch受体的结构。
2、构建上述SynNotch合成受体的表达质粒载体，具体步骤如下所示：
(1)含有热带爪蟾Notch跨膜结构域的SynNotch合成受体(简称SynNotch合成受体1)具有如SEQ ID NO:4所示的氨基酸序列和具有如SEQ ID NO:5所示的核苷酸序列，含有鼠Notch跨膜结构域的SynNotch合成受体(简称SynNotch合成受体2)具有如SEQ ID NO:6所示的氨基酸序列和具有如SEQ ID NO:7所示的核苷酸序列，然后由金唯智公司分别将SEQ ID NO:5所示的核苷酸序列和SEQ ID NO:7所示的核苷酸序列合成至Puc57载体中；
(2)将pCDH-EV载体使用EocR1和BamH1核酸内切酶进行双酶切，使用1％的琼脂糖凝胶电泳20min后，切胶胶回收双酶切载体；
(3)使用引物对从步骤(1)的两个pUC57载体中通过PCR分别得到SEQ ID NO:5所示的核苷酸序列和SEQ ID NO:7所示的核苷酸序列，然后将SEQ ID NO:5所示的核苷酸序列和SEQ ID NO:7所示的核苷酸序列分别与步骤(2)得到的双酶切载体和SynNotch合成受体的核苷酸片段同源重组，得到pCDH-antiGFP-XENTR-SynNotch-Gal4-VP64和pCDH-antiGFP-MOUSE-SynNotch-Gal4-VP64两种表达质粒载体。
(4)将上述构建的两种表达质粒载体(PCDH-antiGFP-XENTR-SynNotch-Gal4VP64和pCDH-antiGFP-MOUSE-SynNotch-Gal4-VP64)进行测序验证，测序结果验证构建成功。

实施例2：SynNotch受体在细胞水平的试验及验证
本实施例中，SynNotch受体细胞识别特定胞外信号的具体过程为：当SynNotch受体的胞外域部分anti-GFP抗体单链结合到传输细胞(sender cell)的GFP抗原后，Notch跨膜结构域被蛋白酶切割，GAL4VP64被释放入胞内，GAL4可以特异识别并结合到GAL4结合位点(UAS区域)，VP64转录激活元件就会激活下游BFP基因的转录，促进BFP荧光蛋白表达。具体参见图2。
本发明以HT1080细胞作为传输细胞(sender cell)，同样以另一批HT1080细胞作为接收细胞(receiver cell)，对实施例1制备的表达质粒载体(pCDH-antiGFP-XENTR-SynNotch-Gal4VP64)增强下游基因转录激活的能力进行细胞水平的验证，具体的验证步骤如下：
(1)HT1080细胞系感染慢病毒，可稳定表达膜锚定的GFP，作为传输细胞(膜锚定的GFP的结构如图3所示，GFP具有如SEQ ID NO:8所示的氨基酸序列以及如SEQ ID NO:9所示的核苷酸序列)。
其中，GFP的氨基酸和核苷酸序列如下所示：

取另一批HT1080细胞作为接收细胞，先感染pHR_Gal4UAS_tBFP_PGK_mCherry慢病毒(购自Addgene，#79130，该慢病毒感染宿主细胞后，可被GAL4VP64诱导表达BFP荧光蛋白)，分选出能稳定高表达pHR_Gal4UAS_tBFP_PGK_mCherry的细胞，然后感染pCDH-antiGFP-XENTR-SynNotch-Gal4VP64慢病毒。
pHR_Gal4UAS_tBFP_PGK_mCherry和pCDH-antiGFP-XENTR-SynNotch-Gal4VP64慢病毒是将表达质粒(pCDH-antiGFP-XENTR-SynNotch-Gal4VP64或pHR_Gal4UAS_tBFP_PGK_mCherry)、pMD2.G和psPAX2共转染进入含密度为90％的HEK293-T细胞的15cm培养皿中，三天后收取细胞上清液，使用0.45μM滤器过滤病毒液并行病毒浓缩即得。
(2)取上述稳定转染的接收细胞HT1080细胞，以细胞密度为3×105个/mL接种到24孔板中，加入细胞密度为3×105个/mL且能表达GFP的HT1080细胞共培养48小时。使用Olympus倒置荧光显微镜拍摄细胞照片，并使用ImageJ分析细胞表达的荧光强度，结果如图4所示。
图4A为荧光显微镜照片比较，其中，MOUSE-Syn表示含有鼠Notch跨膜结构域的SynNotch合成受体(简称SynNotch合成受体2)，即为原始的系统；XENTR-Syn表示含有热带爪蟾Notch跨膜结构域的SynNotch合成受体(简称SynNotch合成受体1)。由图4A可知，与SynNotch合成受体2相比，SynNotch合成受体1可显著地提高了激活的效率。
图4B为ImageJ软件分析HT1080细胞中表达BFP蓝色荧光蛋白的平均荧光强度。由图4B可知，与SynNotch合成受体2(即为MOUSE组)相比，SynNotch合成受体1(即为XENTR组)的激活效率有95.1％的提升，且SynNotch合成受体1(即为XENTR组)的泄露水平下降了56.4％。
由于细胞间的接触存在三种形式，包括贴壁细胞与贴壁细胞间的接触，贴壁细胞与悬浮细胞间的接触、悬浮细胞与悬浮细胞间的接触。三种接触形式也一定程度的反映着体内细胞三种真实的接触环境，因此确认三种细胞接触方式都能够有效激活SynNotch系统十分的关键。而HT1080是一种贴壁细胞，上述的图4A和图4B结果仅能证明在贴壁细胞与贴壁细胞间的接触能够激活SynNotch系统，但是在其他两种接触体系中却不能确定。因此，进一步按照本实施例步骤(1)的方式构建了悬浮的接收细胞Jurkat和悬浮的发射细胞K562。进一步地，按照本实施例步骤(2)的方式，将发射细胞K562和接收细胞Jurkat共培养48小时，观察悬浮细胞与悬浮细胞间的接触能否激活SynNotch系统；同时，也将发射细胞HT1080和接收细胞Jurkat共培养48小时，观察悬浮细胞与贴壁细胞间的接触能否激活SynNotch系统。结果如图4C和图4D所示，不论在悬浮接收细胞与悬浮发射细胞间的接触中，还是悬浮接受细胞与贴壁发射细胞间的接触中，XENTR-SynNotch和MOUSE-SynNotch都能够有效的激活。并且，XENTR-SynNotch的泄露激活效率远低于MOUSE-SynNotch，有效激活效率略高于MOUSE-SynNotch。
因此，可说明本发明设计的含有来源于热带爪蟾(Xenopus tropicalis)的Notch受体蛋白的跨膜区的SynNotch系统比原始含有来源于鼠的Notch受体蛋白的跨膜区的SynNotch受体系统具有更强的激活效率。
实施例3：靶癌细胞对SynNotch-CAR门控嵌合抗原受体联用的Jurkat细胞的激活
目前，以表达嵌合抗原受体(CAR)的T细胞作为某些B细胞癌症的治疗是有效的。然而，CAR-T细胞癌症免疫疗法的一个主要问题是脱靶效应，其中治疗性CAR-T细胞会破坏正常组织，从而导致严重的副作用甚至死亡。为缓解此类问题的可能策略是，使治疗性的CAR-T细胞具有更精确的肿瘤识别靶点，而增加一个肿瘤抗原识别才能使CAR表达从而提供更精确的T细胞应答，是一种有效的手段。为了实现这一策略，推断SynNotch合成受体可用于治疗性CAR-T细胞的设计，即首先通过结合肿瘤特异性细胞表面抗原检测肿瘤并且仅对肿瘤中的第二肿瘤特异性抗原引发CAR的表达，由 此即提供了关于CAR-T细胞活性的双抗原控制，又提供了肿瘤定位性应答。
基于此，发明人设计了SynNotch-CAR门控嵌合抗原受体联用的Jurkat细胞(简称SynNotch-CAR-Jurkat T细胞)含有通过在Jurkat细胞(T细胞系)中进行相应实验给出，具体包括以下步骤：
(1)将实施例2中的SynNotch合成受体1进行慢病毒包装，同时慢病毒包装可以识别CD19的CAR分子，编码该CAR分子的核酸的5’端前包括一段UAS-minimal-CMV序列，该序列可以与SynNotch合成受体激活后产生的胞内多肽结合，诱导该CAR分子的表达(由金唯智公司直接合成并进行慢病毒包装，该CAR分子具有如SEQ ID NO:10所示的氨基酸序列以及如SEQ ID NO:11所示的核苷酸序列，后续称该CAR分子为诱导型CAR分子)。将上述两种病毒同时感染Jurkat T细胞，通过筛选得到稳定表达上述SynNotch合成受体和诱导型CAR分子的Jurkat T细胞；
(2)采用本领域的常规方法构建单独表达膜锚定GFP、单独表达膜锚定CD19(结构如图5所示，CD19具有如SEQ ID NO:12所示的氨基酸序列以及如SEQ ID NO:13所示的核苷酸序列)和共同表达膜锚定GFP和CD19的K562细胞作为传输细胞；
(3)将上述步骤(2)的3种K562细胞分别与步骤(1)中所构建的Jurkat细胞进行共孵育，在24小时后检测各组Jurkat细胞CD69的表达和IL-2、TNF-α的分泌情况，结果参见图6。其中，CD69的表达和IL-2、TNF-α的分泌代表着Jurkat T细胞的激活。
如图6所示，只有当暴露于表达两种抗原(GFP和CD19)的K562时，SynNotch-CAR-Jurkat T细胞才能被激活。

实施例4：SynNotch-CAR门控嵌合抗原受体联用的NK-92细胞(简称SynNotch-CAR-NK-92细胞)对靶癌细胞的杀伤
本发明涉及的SynNotch合成受体1不仅在T细胞中能够发挥作用，在其他类型的免疫细胞中也应该是通用的。发明人通过在NK-92细胞中应用与实施例4中所述相同的SynNotch-CAR系统证实了其在其他免疫细胞中同样能够发挥作用，具体包括以下步骤：
(1)将实施例2中的SynNotch合成受体(SynNotch合成受体1或SynNotch合成受体2)进行慢病毒包装，同时慢病毒包装可以识别CD19的CAR分子，编码该CAR分子的核酸序列的5’的前端包括一段UAS-minimal-CMV序列，该序列可以与SynNotch合成受体激活后产生的胞内多肽结合，诱导该CAR分子的表达(由金唯智公司直接合成并进行慢病毒包装，该CAR分子具有如SEQ ID NO:10所示的氨基酸序列以及如SEQ ID NO:11所示的核苷酸序列)。将上述两种病毒同时感染NK-92细胞，通过筛选得到稳定表达上述SynNotch合成受体和CAR分子的NK-92细胞；同时设置稳定表达的可以识别CD19的CAR分子的NK-92细胞(即为CAR-NK)作为阳性杀伤对照组，NK-EV(感染了空白质粒生产的病毒的NK细胞)作为阴性杀伤对照组；
(2)构建单独表达膜锚定GFP、单独表达膜锚定CD19(具体序列参见实施例3的步骤(2))和共同表达膜锚定GFP和CD19的Huh7细胞作为传输细胞；
(3)将(2)中所述传输细胞用CTV染料标记(购自赛默飞)；
(4)将步骤(3)中的3种Huh7细胞分别与(1)中所构建的NK-92细胞进行共孵育，在48小时后收集细胞，并用PI染料标记死细胞，分析检测各组Huh7细胞的死活情况，具体参见图7。其中，Huh7细胞的死亡比例代表着NK-92细胞对传输细胞的杀伤情况。
如图7所示，当暴露于同时表达两种抗原(GFP和CD19)的Huh7细胞时，SynNotch-CAR-NK-92细胞才能够被显著地激活，发挥出更高的对Huh7细胞的杀伤效应。其中，相较于含有SynNotch合成受体2的SynNotch-CAR(M-Syn-CAR)联用，含有SynNotch合成受体1的SynNotch-CAR(X-Syn-CAR)联用的杀伤效率略微提升；并且，含有SynNotch合成受体1的SynNotch-CAR联用的NK-92细胞杀伤效率与稳定表达可以识别CD19的CAR分子(CAR-NK)的NK-92细胞相比，没有显著差异。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指 结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (31)

  1. 分离的多肽在制备SynNotch合成受体中的用途，所述分离的多肽与来源于热带爪蟾(Xenopus tropicalis)的Notch受体蛋白的跨膜区具有至少80％的同一性。
  2. 一种嵌合多肽，其特征在于，包括：
    胞外区，所述胞外区具有结合第一分子活性；
    跨膜区，所述跨膜区的N端与所述胞外区的C端相连；
    胞内区，所述胞内区的N端与所述跨膜区的C端相连；
    其中，所述跨膜区包括分离的多肽，所述分离的多肽与来源于热带爪蟾的Notch受体蛋白的跨膜区具有至少80％的同一性。
  3. 根据权利要求1所述的用途或权利要求2所述的嵌合多肽，其特征在于，所述分离的多肽与来源于热带爪蟾(Xenopus tropicalis)的Notch受体蛋白的跨膜区具有100％的同一性。
  4. 根据权利要求1所述的用途或权利要求2所述的嵌合多肽，其特征在于，所述来源于热带爪蟾(Xenopus tropicalis)的Notch受体蛋白的跨膜区具有如SEQ ID NO:1所示的氨基酸序列。
  5. 根据权利要求2所述的嵌合多肽，其特征在于，所述第一分子包括肿瘤抗原、病毒、细菌、内毒素、抗体、细胞受体和细胞受体的配体中的至少之一。
  6. 根据权利要求5所述的嵌合多肽，其特征在于，所述肿瘤抗原包括肿瘤相关抗原和肿瘤特异性抗原中的至少之一，优选为肿瘤特异性抗原。
  7. 根据权利要求2所述的嵌合多肽，其特征在于，所述第一分子包括但不限于GFP、eGFP、CD19、ALPPL2、BCMA、SIRPα、CD1a、CD1b、CD1c、CD1d、CD1e、CD2、CD3d、CD3e、CD3g、CD4、CD5、CD7、CD8a、CD8b、CD20、CD21、CD22、CD23、CD25、CD27、CD28、CD30、CD33、CD34、CD38、CD40、CD44、CD44v6、CD45、CD48、CD51、CD52、CD56、CD59、CD66、CD70、CD71、CD72、CD73、CD74、CD79A、CD79B、CD80、CD86、CD94、CD95、CD133、CD134、CD140、CD152、CD154、CD158、CD178、CD181、CD182、CD183、CD200、CD210、CD221、CD246、CD252、CD253、CD261、CD262、CD273、CD274、CD276、CD279、CD295、CD339、CD340、EGFR、HER2、FGFR2、AFP、CA125、MSLN、GPC3、CEA、CLDN18.2、EpCAM、PSCA、GD2、IL-13、IL-13RA2、ROR1、MUC 1、PSMA、MAGEA1、4-1BB、5T4、BAFF、CA242、CA-IX、MET、CCR4、CNTO888、FAP、MORAb-009、VEGF-A、VEGFR-1和VEGFR-2中的至少之一。
  8. 根据权利要求2所述的嵌合多肽，其特征在于，所述胞外区包括结合所述第一分子的第一结合蛋白或其片段。
  9. 根据权利要求8所述的嵌合多肽，其特征在于，所述第一结合蛋白或其片段包括抗体或其功能性片段、受体、受体的配体和细胞黏附分子中的至少之一。
  10. 根据权利要求8所述的嵌合多肽，其特征在于，所述第一结合蛋白或其片段为结合所述第一分子的单抗或多抗。
  11. 根据权利要求10所述的嵌合多肽，其特征在于，所述单抗包括Fab抗体、F(ab')2片段、Fv抗体、单链抗体、单域抗体以及最小识别单位的至少之一。
  12. 根据权利要求2所述的嵌合多肽，其特征在于，所述胞内区包括转录激活蛋白、转录阻遏蛋白、转录因子、位点特异性核酸酶、重组酶、活化性免疫受体胞内结构域和抑制性免疫受体胞内结构域中的至少之一。
  13. 根据权利要求12所述的嵌合多肽，其特征在于，所述胞内区包括GaL4 VP64、GaL4 VP16、tetR VP64、ZFHD1 VP64、Gal4 KRAB、HAP1 VP16、LexA-VP64、Cas9和Cas13中的至少之一。
  14. 根据权利要求13所述的嵌合多肽，其特征在于，所述Gal4 VP64具有如SEQ ID NO:2所示的氨基酸序列。
  15. 根据权利要求2所述的嵌合多肽，其特征在于，所述跨膜区和胞内区具有如SEQ ID NO:3所示的氨基酸序列；和/或
    所述嵌合多肽具有如SEQ ID NO:4所示的氨基酸序列。
  16. 一种第一核酸分子，其特征在于，所述第一核酸分子编码权利要求2～15任一项所述的嵌合多肽；
    所述核酸分子为DNA。
  17. 一种第一表达载体，其特征在于，携带权利要求16所述的第一核酸分子。
  18. 根据权利要求17所述的第一表达载体，其特征在于，所述第一表达载体为真核表达载体、原核表达载体、病毒或噬菌体，优选为质粒表达载体。
  19. 一种重组细胞，其特征在于，包括：
    携带权利要求16所述的第一核酸分子或权利要求17～18任一项所述的第一表达载体；或，
    表达权利要求2～15任一项所述的嵌合多肽。
  20. 根据权利要求19所述的重组细胞，其特征在于，所述重组细胞是通过将权利要求17～18任一项所述的第一表达载体引入至宿主细胞中而获得的；
    所述宿主细胞包括免疫细胞、神经元、祖细胞或前体细胞、上皮细胞、内皮细胞和干细胞中的至少之一。
  21. 根据权利要求20所述的重组细胞，其特征在于，所述宿主细胞包括T细胞、B细胞、单核细胞、NK细胞、树突细胞、巨噬细胞、调节T细胞、辅助T细胞、细胞毒性T细胞、NKT细胞和γδT细胞中的至少之一。
  22. 根据权利要求19所述的重组细胞，其特征在于，所述重组细胞进一步包括：
    编码嵌合抗原受体、NK细胞受体或T细胞受体的第二核酸分子或携带所述第二核酸分子的第二表达载体；或表达所述嵌合抗原受体、NK细胞受体或T细胞受体。
  23. 根据权利要求22所述的重组细胞，其特征在于，所述嵌合多肽的胞内区包括转录激活蛋白，编码所述嵌合抗原受体、NK细胞受体或T细胞受体的第二核酸分子的5’端与诱导表达核酸序列相连，所述诱导表达核酸序列用于结合所述转录激活蛋白。
  24. 一种药物组合物，其特征在于，包括权利要求2～15任一项所述的嵌合多肽、权利要求16所述的第一核酸分子、权利要求17～18任一项所述的第一表达载体或权利要求19～23任一项所述的重组细胞；
    以及任选地进一步包括药学上可接受的辅料。
  25. 权利要求2～15任一项所述的嵌合多肽、权利要求16所述的第一核酸分子、权利要求17～18任一项所述的第一表达载体、权利要求19～23任一项所述的重组细胞或权利要求24所述的药物组合物在制备药物中用途，所述药物用于预防和/或治疗疾病；
    任选地，所述疾病包括癌症或肿瘤、自身免疫性疾病、炎症及由细胞衰老所引发的相关疾病。
  26. 权利要求2～15任一项所述的嵌合多肽、权利要求16所述的第一核酸分子、权利要求17～18任一项所述的第一表达载体、权利要求19～23任一项所述的重组细胞或权利要求24所述的药物组合物，用于预防和/或治疗疾病；
    任选地，所述疾病包括癌症或肿瘤、自身免疫性疾病、炎症及由细胞衰老所引发的相关疾病。
  27. 一种预防和/或治疗疾病的方法，其特征在于，包括：
    向受试者施用药学上可接受剂量的权利要求2～15任一项所述的嵌合多肽、权利要求16所述的第一核酸分子、权利要求17～18任一项所述的第一表达载体、权利要求19～23任一项所述的重组细胞或权利要求24所述的药物组合物；
    任选地，所述疾病包括癌症或肿瘤、自身免疫性疾病、炎症及由细胞衰老所引发的相关疾病。
  28. 一种激活免疫细胞的方法，其特征在于，包括：
    将所述免疫细胞与第一分子进行第一接触，其中，所述免疫细胞表达权利要求2～15任一项所述的嵌合多肽。
  29. 根据权利要求28所述的方法，其特征在于，所述嵌合多肽的胞内区包括转录激活蛋白，所述免疫细胞表达嵌合抗原受体、NK细胞受体或T细胞受体，所述嵌合抗原受体、NK细胞受体或T细胞受体包括结合预定抗原的抗体或其功能性片段，编码所述嵌合抗原受体、NK细胞受体或T细胞受体的第二核酸分子的5’端与诱导表达核酸序列相连，所述诱导表达核酸序列用于结合所述转录激活蛋白；
    所述方法进一步包括：
    所述第一接触之后，所述免疫细胞释放所述转录激活蛋白，并表达所述嵌合抗原受体、NK细胞受体或T细胞受体；
    将所述免疫细胞的嵌合抗原受体、NK细胞受体或T细胞受体与所述预定抗原结合，以便激活所述免疫细胞；
    任选地，所述免疫细胞包括T细胞、B细胞、单核细胞、NK细胞、树突细胞、巨噬细胞、调节T细胞、辅助T细胞、细胞毒性T细胞、NKT细胞和γδT细胞中的至少之一。
  30. 一种追踪第一细胞-第二细胞接触的方法，其特征在于，包括：
    将所述第一细胞和所述第二细胞进行第二接触，其中，所述第一细胞表达权利要求2～15任一项所述的嵌合多肽和报告基因蛋白，所述嵌合多肽的胞内区为转录激活蛋白，编码所述报告基因蛋白的第三核酸分子的5’端与诱导表达核酸序列相连，所述诱导表达核酸序列用于结合所述转录激活蛋白，所述第二细胞表达第一分子；
    基于所述第一细胞中所述报告基因蛋白的检测结果，确定第一细胞和第二细胞的接触情况。
  31. 根据权利要求30所述的方法，其特征在于，所述第一细胞或第二细胞包括免疫细胞、神经元、祖细胞或前体细胞、上皮细胞、内皮细胞和干细胞中的至少之一；
    任选地，所述第一细胞包括T细胞、B细胞、单核细胞、NK细胞、树突细胞、巨噬细胞、调节T细胞、辅助T细胞、细胞毒性T细胞、NKT细胞和γδT细胞中的至少之一。
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