SynNotch receptors and their uses

Abstract

The present invention relates to SynNotch receptors and uses thereof, and specifically proposes 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 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 transcription of downstream genes, and reduce the background leakage of the chimeric polypeptide.

Description

Technical Field

[0001] 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. Background Art

[0002] With the development of synthetic biology, it has become a reality to transform cells so that they can recognize specific extracellular signals and transmit the recognized signals in the transformed cells to make artificially designed responses, and it has gradually been applied in the field of biomedicine. For example, the application of chimeric antigen receptor (CAR) technology in clinical treatment. In addition, in 2016, Wendell A. Lim's team developed a SynNotch receptor system based on the Notch signaling pathway. The system can also recognize cell membrane surface ligands, and its signal output is not limited to simple immune cell activation signals. It can achieve a variety of different signal output types, such as activation and inhibition of specific gene expression. This 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.

[0003] In biology, the traditional Notch signaling pathway (system) includes Notch corresponding ligands, Notch molecules and Notch-regulated downstream genes. The Notch molecule is a transmembrane molecule, which can be divided into three parts: extracellular structure, transmembrane domain and intracellular domain. After the extracellular domain binds to the corresponding Notch ligand, 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 γ-secretase and released into the cell, and then enters the nucleus as a transcription factor to regulate the expression of downstream related genes. Wendell A. Lim's team transformed the Notch1 receptor of mice, replacing its extracellular domain with a single-chain antibody or nano-antibody, and replacing the intracellular domain with a transcriptional activation or transcriptional inhibition domain, retaining only the transmembrane domain that can be recognized and cut by the protease, and also equipped with downstream regulated gene elements. For different targets, 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. By changing the gene elements regulated downstream of SynNotch, 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. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a SynNotch receptor, which can enhance its ability to activate downstream genes and reduce the background leakage activation level.

[0005] The present invention is accomplished based on the following findings of the inventors:

[0006] 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.

[0007] However, 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. Taking into account the differences in the S2 and S3 cleavage sequences carried on the Notch transmembrane domains of different species, as well as the conformational and sequence preferences of the S2 and S3 cleavage enzymes from different species, 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 inventor took the transformation of human cells with the SynNotch synthetic receptor as the ultimate goal. During the experiment, he accidentally discovered that selecting the Notch transmembrane domain of tropical clawed frogs can improve the ability and efficiency of the SynNotch synthetic receptor to activate downstream gene transcription, as well as reduce the background leakage activation of the SynNotch synthetic receptor, so that the SynNotch synthetic receptor can play a more effective role.

[0008] Therefore, in one aspect of the present invention, 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.

[0009] In another aspect of the present invention, a chimeric polypeptide is proposed. According to an embodiment of the present invention, the chimeric polypeptide 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 transcription of downstream genes, and reduce the background leakage of the chimeric polypeptide.

[0010] In another aspect of the present invention, the present invention provides a first nucleic acid molecule. According to an embodiment of the present invention, the first nucleic acid molecule encodes the aforementioned chimeric polypeptide of claim 1. According to an embodiment of the present invention, the first nucleic acid molecule encodes the aforementioned chimeric polypeptide.

[0011] In another aspect of the present invention, the present invention provides a first expression vector. According to an embodiment of the present invention, 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.

[0012] In another aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, 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.

[0013] In another aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes the aforementioned chimeric polypeptide, the aforementioned first nucleic acid molecule, the aforementioned first expression vector or the aforementioned recombinant cell. As can be seen from the above, 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 for the treatment of cancer, etc.

[0014] In another aspect of the present invention, 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. As mentioned above, 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. Thus, 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.

[0015] In another aspect of the present invention, the present invention provides a method for activating immune cells. According to an embodiment of the present invention, the method comprises: first contacting the immune cell with a first molecule, wherein the immune cell expresses the aforementioned chimeric polypeptide. Thus, 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.

[0016] In another aspect of the present invention, the present invention proposes a method for tracking the contact between a first cell and a second cell. According to an embodiment of the present invention, 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. Thus, 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.

[0017] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0019] Figure 1 is a schematic diagram of the structure of the SynNotch synthetic receptor in Example 1 of the present invention;

[0020] Figure 2 A schematic diagram of SynNotch synthetic receptor cells recognizing specific extracellular signals in Example 2 of the present invention;

[0021] Figure 3 This is a schematic diagram of the structure of membrane-anchored GFP in Example 2 of the present invention;

[0022] Figure 4 The leakage efficiency and effective activation efficiency of the SynNotch synthetic receptor containing the mouse Notch transmembrane domain and the SynNotch synthetic receptor containing the tropical clawed frog Notch transmembrane domain in Example 2 of the present invention;

[0023] Figure 5 This is a schematic diagram of the structure of membrane-anchored CD19 in Example 3 of the present invention;

[0024] Figure 6 The activation result of SynNotch-CAR-Jurkat T cells by target cancer cells in Example 3 of the present invention;

[0025] Figure 7 This is the killing efficiency of SynNotch-CAR-NK 92 cells on target cancer cells in Example 4 of the present invention. DETAILED DESCRIPTION

[0026] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0027] It should be noted that the terms "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.

[0028] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0029] As used herein, 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.

[0030] As used herein, the terms "identity", "homology" or "similarity" are used to describe an amino acid sequence or a 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 (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 48:443); the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:24 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0031] As used herein, 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.

[0032] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and / or between host cells. The expression vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for the 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 can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0033] In this article, 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. Among them, 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 nucleic acids (such as vectors) into cells 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 the target protein.

[0034] As used herein, 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.

[0035] In this article, 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.

[0036] As used herein, 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. Preferably, the composition of the present invention is administered by intravenous or subcutaneous injection.

[0037] As used herein, the term "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" as used herein 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" as used herein 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.

[0038] 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.

[0039] Use of isolated polypeptides in the preparation of SynNotch synthetic receptors

[0040] In one aspect of the present invention, 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.

[0041] According to an embodiment of the present invention, the isolated polypeptide has 100% identity with the transmembrane region of the Notch receptor protein derived from Xenopus tropicalis.

[0042] According to an embodiment of the present invention, the transmembrane region of the Notch receptor protein derived from Xenopus tropicalis has an amino acid sequence as shown in SEQ ID NO:1.

[0043] ILDYGFIGLGGKNITPPDNEEICENEQCAELADNKICNANCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFNDGKCDSQCNNSGCLYDGFDCQKVEVQCNPLYDQYCRDHFQDGHCDQGCNNAECEWDGLDCDNMPENLAEGTLLIVVLMPPEKLKNNSVNFLRE LSRVLHTNVVFKKDSKGEYKIYPYYGNEEELKKHHIKKRSAASWSDAPTAIFSTMKESVLPGRRRRELDQMEVRGSIVYLEIDNRQCYKSSSQCFTSATDVAAFLGALATHGNLNIPYKIEAVKSEIVETAKPPPPLYAMFSMLVIPLLIIFVIMVVIVNKKRRR(SEQ IDNO:1).

[0044] Chimeric polypeptide

[0045] In another aspect of the present invention, the present invention provides a chimeric polypeptide. According to an embodiment of the present invention, 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.

[0046] 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 the transcription of downstream genes, and reduce the background leakage of the chimeric polypeptide. In addition, the cells expressing the chimeric polypeptide can recognize the first molecule, and after binding to the first molecule, they can achieve a variety of different signal output types, such as activation and inhibition of the expression of specific genes, and are used to prepare immune cells expressing the chimeric polypeptide. After contacting the first molecule, the immune cells can secrete factors with therapeutic effects (such as single-chain antibodies, cytokines, etc.) for anti-tumor.

[0047] According to an embodiment of the present invention, the isolated polypeptide has 100% identity with the transmembrane region of the Notch receptor protein derived from Xenopus tropicalis.

[0048] According to an embodiment of the present invention, the transmembrane region of the Notch receptor protein derived from Xenopus tropicalis has an amino acid sequence as shown in SEQ ID NO:1.

[0049] According to an embodiment of the present invention, the transmembrane region further comprises an epidermal growth factor-like repeat sequence (EGFrepeat) and / or a RAM sequence, thereby further reducing the background leakage activation of the SynNotch synthetic receptor.

[0050] According to an embodiment of the present invention, the epidermal growth factor-like repeat sequence is an amino acid sequence as shown in SEQ ID NO:15.

[0051] VVSPCASRPCYNGGTCQFSPEEPFFQCFCPTNFNGLFCH (SEQ ID NO: 15).

[0052] According to an embodiment of the present invention, the RAM sequence is the amino acid sequence shown in SEQ ID NO:16.

[0053] EHGQLWFP (SEQ ID NO: 16).

[0054] According to an embodiment of the present invention, 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.

[0055] According to an embodiment of the present invention, 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.

[0056] In this article, the term "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. Among them, tumor-specific antigens (TSA) are new antigens that are unique to tumor cells or only exist in certain tumor cells but not in normal cells. 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. Among them, they include but are not limited to PD-L1, PD-1, TGF-β, CEA, GD2 and GD3.

[0057] In this article, the term "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), immunomodulatory receptors (including positive co-receptors (such as CD28) and negative (immunosuppressive) co-receptors (such as PD1)) and receptors for immunomodulatory molecules (such as TGFβ), etc.

[0058] In this article, the term "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.

[0059] Exemplarily, the ligand is VEGF and the receptor is VEGF receptor; or the ligand is heregulin and the receptor is Her2.

[0060] In this article, the term "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.

[0061] In a preferred embodiment of the present invention, the tumor antigen is a tumor-specific antigen.

[0062] According to an embodiment of the present invention, the first molecule includes 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, CD79 B. 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. At least one of PSMA, MAGEA1, 4-1BB, 5T4, BAFF, CA242, CA-IX, MET, CCR4, CNTO888, FAP, MORAb-009, VEGF-A, VEGFR-1 and VEGFR-2.

[0063] According to an embodiment of the present invention, the extracellular region includes a first binding protein or a fragment thereof that binds to the first molecule;

[0064] According to an embodiment of the present invention, 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.

[0065] In this article, "cell adhesion molecule" or "CAM" can refer to a polypeptide that binds to a component of the extracellular matrix (ECM) or a cell surface molecule. For example, the cell adhesion molecule can be the extracellular region of the CAM, wherein the CAM can be a calcium-independent adhesion molecule, such as the CAM is an immunoglobulin superfamily CAM, and the CAM can also be a calcium-dependent adhesion molecule, such as the CAM is an integrin, a cadherin or a selectin; the cell adhesion molecule can be an integrin, such as a cadherin, such as E-cadherin, P-cadherin, N-cadherin, R-cadherin, M-cadherin, etc.; the cell adhesion molecule can also be a selectin, such as E-selectin, L-selectin or P-selectin.

[0066] In this article, 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. Among them, 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.

[0067] 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).

[0068] In this article, 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 the antigen. For example, the fragment may comprise part or all of the antibody CDR. Such fragments are biologically active because they bind to the 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).

[0069] According to an embodiment of the present invention, the first binding protein or fragment thereof is a monoclonal antibody or a polyclonal antibody that binds to the first molecule.

[0070] In this article, the terms "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.

[0071] According to an embodiment of the present invention, 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.

[0072] In this article, the terms "single domain antibody", "nanoantibody" and "VHH antibody" are used interchangeably, which were originally described as antigen-binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), comprising a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind to antigen proteins (e.g., D-dimers) through the heavy chain variable region.

[0073] As used herein, the term "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.

[0074] As used herein, the term "F(ab')2 antibody" or "F(ab')2 fragment" has two antigen-binding F(ab') portions linked together by a disulfide bond.

[0075] As used herein, the term "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.

[0076] As used herein, the terms "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.

[0077] In this article, the terms "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.

[0078] According to an embodiment of the present invention, 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.

[0079] According to an embodiment of the present invention, the intracellular region includes at least one of GaL4VP64, GaL4 VP16, tetR VP64, ZFHD1VP64, Gal4KRAB, HAP1VP16, LexA-VP64, Cas9 and Cas13.

[0080] According to an embodiment of the present invention, the Gal4VP64 has an amino acid sequence as shown in SEQ ID NO:2.

[0081] MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSAAAGGSGGSGGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDFDFDLDMLGS(SEQ IDNO:2).

[0082] According to an embodiment of the present invention, the transmembrane region and the intracellular region have an amino acid sequence as shown in SEQ ID NO:3.

[0083] VVSPCASRPCYNGGTCQFSPEEPFFQCFCPTNFNGLFCHILDYGFIGGLGKNITPPDNEEICENEQCAELADNKICNANCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFNDGKCDSQCNNSGCLYDGFDCQKVEVQCNPLYDQYCRDHFQDGHCDQGCNNAECEWDGLDCDNMPENLAEGTLLIVVLMPPEKLKNNSVNFLRELSRVLHTNVVFKKDSKGEYKIYPYYGNEEELKKHHIKKRSAASWSDAPTAIFSTMKESVLPGRRRRELDQMEVRGSIVYLEIDNRQCYKSSSQCFTSATDVAAFLGALATHGNLNIPYKIEAVKSEIVETAKPPPPLYAMFSMLVIPLLIIFVIMVVIVNKKRRREHGQLWFPMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSAAAGGSGGSGGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGS(SEQ ID NO:3).

[0084] According to an embodiment of the present invention, the chimeric polypeptide has the amino acid sequence shown in SEQ ID NO:4.

[0085] (SEQ ID NO:4).

[0086] Nucleic acid molecules, vectors, recombinant cells and pharmaceutical compositions

[0087] In another aspect of the present invention, the present invention provides a first nucleic acid molecule. According to an embodiment of the present invention, 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.

[0088] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0089] It should be noted that, for 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. In addition, 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.

[0090] In another aspect of the present invention, the present invention provides a first expression vector. According to an embodiment of the present invention, the first expression vector carries the aforementioned first nucleic acid molecule. When the above-mentioned first nucleic acid molecule is connected to the vector, 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. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Of course, the first nucleic acid molecule can be operably connected to the control element.

[0091] Herein, "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. 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.

[0092] According to an embodiment of the present invention, the first expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0093] According to an embodiment of the present invention, the first expression vector is a plasmid expression vector.

[0094] In another aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present invention, 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.

[0095] It should be noted that the "suitable conditions" described in the present invention 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.

[0096] According to an embodiment of the present invention, the recombinant cell is obtained by introducing the aforementioned first expression vector into a host cell.

[0097] According to an embodiment of the present invention, the host 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.

[0098] According to an embodiment of the present invention, 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 γδT cells.

[0099] According to an embodiment of the present invention, 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. 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). It is found that 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.).

[0100] As used herein, the term "chimeric antigen receptor (CAR)" 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. "Chimeric Antigen Receptor (CAR)" is also referred to as "chimeric receptor", "T-body" or "chimeric immune receptor (CIR)". The "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide capable of binding to an antigen. "Intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain that transmits signals to activate or inhibit intracellular biological processes.

[0101] Herein, the term "T cell receptor (abbreviated as TCR)" 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 (α) and beta (β) chains in about 95% of T cells, while about 5% of T cells have TCRs composed of gamma (γ) and delta (δ) chains. During antigen processing, the antigen is degraded within the cell and then carried to the cell surface by the major histocompatibility complex (MHC) molecule. 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. Among them, 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). The human leukocyte antigen system (HLA) is the name of the gene complex that encodes the human major histocompatibility complex (MHC), and includes HLA class I antigens (A, B, and C) and HLA class II antigens (DP, DQ, and DR). HLA alleles A, B, and C present peptides that are primarily derived from intracellular proteins, such as proteins expressed within cells.

[0102] Herein, the term "NK cell receptor" is 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. Among them, 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.

[0103] According to an embodiment of the present invention, 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, which is used to bind to the transcriptional activator protein.

[0104] Exemplarily, the intracellular region is GaL4VP64, and the induced expression nucleic acid sequence is a UAS-minimal-CMV sequence.

[0105] The UAS-minimal-CMV sequence has a nucleic acid sequence as shown in SEQ ID NO:14.

[0106] GGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGGAGCATGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGA CTAGTTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGC(SEQ IDNO:14).

[0107] In an optional embodiment of the present invention, the chimeric antigen receptor, NK cell receptor or T cell receptor has the activity of binding to a second molecule. For example, the chimeric antigen receptor binds to the second molecule mainly through its extracellular region.

[0108] According to an embodiment of the present invention, 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.

[0109] In an optional embodiment of the present invention, the second molecule includes 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, CD 79B, CD80, CD86, CD94, CD95, CD133, CD134, CD140, CD152, CD154, CD158, CD178, CD181, CD182, CD183, CD200, CD210, CD221, CD246, CD252, CD253, CD261, CD26 2. 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. At least one of PSMA, MAGEA1, 4-1BB, 5T4, BAFF, CA242, CA-IX, MET, CCR4, CNTO888, FAP, MORAb-009, VEGF-A, VEGFR-1 and VEGFR-2.

[0110] In another aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes the aforementioned chimeric polypeptide, the aforementioned first nucleic acid molecule, the aforementioned first expression vector or the aforementioned recombinant cell. As can be seen from the above, 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, etc.

[0111] According to an embodiment of the present invention, it further includes a pharmaceutically acceptable excipient.

[0112] use

[0113] In another aspect of the present invention, 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. As can be seen from the above, 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 with a therapeutic effect (such as a single-chain antibody, a cytokine, 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 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. Thus, 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.

[0114] According to an embodiment of the present invention, the disease includes cancer or tumor, and immune-related disease.

[0115] According to an embodiment of the present invention, the disease includes cancer or tumor, autoimmune disease, inflammation and related diseases caused by cell aging.

[0116] In this article, the term "cancer" or "tumor" can be any unregulated cell growth. Exemplarily, 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.

[0117] method

[0118] In another aspect of the present invention, the present invention provides a method for activating immune cells. According to an embodiment of the present invention, the method comprises: first contacting the immune cell with a first molecule, wherein the immune cell expresses the aforementioned chimeric polypeptide. Thus, 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 performing in vitro cell culture on the immune cell to obtain the activated immune cell.

[0119] According to an embodiment of the present invention, the intracellular region of the chimeric polypeptide includes a transcriptional activator protein, the immune cell expresses a chimeric antigen receptor, a 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, and 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 a predetermined antigen to activate the immune cell.

[0120] According to an embodiment of the present invention, the inducible expression nucleic acid sequence has a nucleic acid sequence as shown in SEQ ID NO:14.

[0121] According to an embodiment of the present invention, the first molecule is a tumor antigen.

[0122] In an optional embodiment of the present invention, the first molecule includes 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, CD 79B, CD80, CD86, CD94, CD95, CD133, CD134, CD140, CD152, CD154, CD158, CD178, CD181, CD182, CD183, CD200, CD210, CD221, CD246, CD252, CD253, CD261, CD26 2. 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. At least one of PSMA, MAGEA1, 4-1BB, 5T4, BAFF, CA242, CA-IX, MET, CCR4, CNTO888, FAP, MORAb-009, VEGF-A, VEGFR-1 and VEGFR-2.

[0123] According to an embodiment of the present invention, 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 γδT cells.

[0124] In another aspect of the present invention, the present invention proposes a method for tracking the contact between a first cell and a second cell. According to an embodiment of the present invention, 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, the contact between the first cell and the second cell is determined. Thus, 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.

[0125] According to an embodiment of the present invention, the inducible expression nucleic acid sequence has a nucleic acid sequence as shown in SEQ ID NO:14.

[0126] According to an embodiment of the present invention, 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.

[0127] According to an embodiment of the present invention, the first molecule is a tumor antigen.

[0128] In an optional embodiment of the present invention, the first molecule includes 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, CD 79B, CD80, CD86, CD94, CD95, CD133, CD134, CD140, CD152, CD154, CD158, CD178, CD181, CD182, CD183, CD200, CD210, CD221, CD246, CD252, CD253, CD261, CD26 2. 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. At least one of PSMA, MAGEA1, 4-1BB, 5T4, BAFF, CA242, CA-IX, MET, CCR4, CNTO888, FAP, MORAb-009, VEGF-A, VEGFR-1 and VEGFR-2.

[0129] According to an embodiment of the present invention, 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.

[0130] According to an embodiment of the present invention, 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 γδT cells.

[0131] In another aspect of the present invention, the present invention provides a method for regulating cell activity. According to an embodiment of the present invention, the method comprises: contacting the cell with a first molecule for a third time, wherein the cell expresses the aforementioned chimeric polypeptide.

[0132] According to an embodiment of the present invention, 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.

[0133] According to an embodiment of the present invention, 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 γδT cells.

[0134] In another aspect of the present invention, the present invention proposes a method for promoting the expression of a gene or protein in a cell. According to an embodiment of the present invention, 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.

[0135] According to an embodiment of the present invention, 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, the hormone, the chemokine or the antibody comprises a second binding protein of the transcription activator protein or a fragment thereof.

[0136] According to an embodiment of the present invention, 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.

[0137] According to an embodiment of the present invention, 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 γδT cells.

[0138] In another aspect of the present invention, the present invention proposes a method for treating or preventing a disease. According to an embodiment of the present invention. The method comprises: administering a pharmaceutically acceptable amount of the aforementioned recombinant cell or the aforementioned pharmaceutical composition to a subject. As can be seen from the foregoing, 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. Thus, the drug of the recombinant cell or pharmaceutical composition containing the chimeric polypeptide can target the first molecule for the prevention and / or treatment of related diseases, such as cancer, etc.

[0139] 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.

[0140] 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). These drugs can be prepared in various forms. For example, 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.

[0141] According to an embodiment of the present invention, the administration route of the method is subcutaneous injection or intravenous injection.

[0142] According to an embodiment of the present invention, the disease includes cancer or tumor, immune-related disease. According to an embodiment of the present invention, the disease includes cancer or tumor, autoimmune disease, inflammation and related diseases caused by cell senescence.

[0143] According to an embodiment of the present invention, 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.

[0144] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0145] Example 1: Design of SynNotch synthetic receptor and construction of its expression plasmid vector

[0146] 1. The structure of the SynNotch synthetic receptor (referred to as the SynNotch receptor) includes an extracellular domain (also referred to as the extracellular region), a Notch transmembrane domain (also referred to as the transmembrane region of the Notch receptor protein, referred to as the transmembrane region) and an intracellular domain (also referred to 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. 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 modified SynNotch receptor designed by the present invention is as follows Figure 1 shown. Figure 1 The structures of SynNotch receptors containing the Notch transmembrane domain (Notch core) of mouse or Xenopus tropicalis are shown.

[0147] 2. Construct the expression plasmid vector of the above-mentioned SynNotch synthetic receptor. The specific steps are as follows:

[0148] (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 the nucleotide sequence shown in SEQ ID NO:5 and the nucleotide sequence shown in SEQ ID NO:7 are synthesized into the Puc57 vector by GeneWei Corporation;

[0149] (2) 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;

[0150] (3) Using primer pairs, the nucleotide sequence shown in SEQ ID NO:5 and the nucleotide sequence shown in SEQ ID NO:7 are 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 are 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.

[0151] (4) The two expression plasmid vectors constructed above (PCDH-antiGFP-XENTR-SynNotch-Gal4VP64 and pCDH-antiGFP-MOUSE-SynNotch-Gal4-VP64) were sequenced and verified, and the sequencing results confirmed that the construction was successful.

[0152]

[0153] MALPVTALLLPLALLLHAARPEQKLISEEDLMADVQLVESGGGLVQAGGSLRLSCAASGRTISMAAMSWFRQAPGKEREFVAGISRSAGSAVHADSVKGRFTISRDNTKNTLYLQMNSLKAEDTAVYYCAVRTSGFFGSIPRTGTAFDYWGQGTQVTVSILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRRMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSAAAGGSGGSGGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGS(SEQ ID NO:6);

[0154]

[0155] Example 2: Testing and verification of SynNotch receptors at the cellular level

[0156] In this embodiment, 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 BFP fluorescent protein. For details, see Figure 2 .

[0157] 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:

[0158] (1) HT1080 cell line was infected with lentivirus, which can stably express membrane-anchored GFP and serve as a delivery cell (the structure of membrane-anchored GFP is as follows Figure 3 As shown, GFP has the amino acid sequence shown in SEQ ID NO:8 and the nucleotide sequence shown in SEQ ID NO:9).

[0159] The amino acid and nucleotide sequences of GFP are as follows:

[0160] METDTLLLWVLLLWVPGSTGDMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGN ILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR(SEQ ID NO:8);

[0161] ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCCGGCAGCACCGGCGACATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGAACGCCGTGGGCCAGGACACCCAGGAGGTGATCGTGGTGCCCCACAGCCTGCCCTTCAAGGTGGTGGTGATCAGCGCCATCCTGGCCCTGGTGGTGCTGACCATCATCAGCCTGATCATCCTGATCATGCTGTGGCAGAAGAAGCCCCGC(SEQ ID NO:9)。

[0162] Another batch of 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.

[0163] The 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 μM filter, and the virus was concentrated.

[0164] (2) Take the above stably transfected recipient cells HT1080 cells and use a cell density of 3×10 5 The cells were inoculated into 24-well plates at a density of 3 × 10 5 HT1080 cells expressing GFP were co-cultured for 48 hours. Cells were photographed using an Olympus inverted fluorescence microscope, and the fluorescence intensity of the cells was analyzed using ImageJ. The results are shown in the figure. Figure 4 shown.

[0165] Figure 4 A is a comparison of fluorescence microscope photos, where MOUSE-Syn represents the synthetic SynNotch receptor containing the mouse Notch transmembrane domain (referred to as SynNotch synthetic receptor 2), which is the original system; XENTR-Syn represents the synthetic SynNotch receptor containing the tropical clawed frog Notch transmembrane domain (referred to as SynNotch synthetic receptor 1). Figure 4 A shows that compared with SynNotch synthetic receptor 2, SynNotch synthetic receptor 1 can significantly improve the activation efficiency.

[0166] Figure 4 B is the average fluorescence intensity of BFP blue fluorescent protein expressed in HT1080 cells analyzed by ImageJ software. Figure 4B shows that compared with SynNotch synthetic receptor 2 (ie, MOUSE group), the activation efficiency of SynNotch synthetic receptor 1 (ie, XENTR group) was increased by 95.1%, and the leakage level of SynNotch synthetic receptor 1 (ie, XENTR group) was decreased by 56.4%.

[0167] Since there are three forms of cell-to-cell contact, including contact between adherent cells, contact between adherent cells and suspended cells, and contact between suspended cells. The three forms of contact also reflect the three real contact environments of cells in the body to a certain extent. Therefore, it is very important to confirm that all three cell contact methods can effectively activate the SynNotch system. HT1080 is an adherent cell. Figure 4 A and Figure 4 Result B can only prove that the contact between adherent cells can activate the SynNotch system, but it cannot be confirmed in the other two contact systems. Therefore, suspended receiving cells Jurkat and suspended transmitting cells K562 were constructed in accordance with the method of step (1) of this embodiment. Further, in accordance with the method of step (2) of this embodiment, the transmitting cells K562 and the receiving cells Jurkat were co-cultured for 48 hours to observe whether the contact between suspended cells and 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 suspended cells and adherent cells can activate the SynNotch system. The results are as follows. Figure 4 C and Figure 4 As shown in D, 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. In addition, 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.

[0168] Therefore, it can be explained that the SynNotch system designed by the present invention containing the transmembrane region of the Notch receptor protein derived from 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.

[0169] Example 3: Activation of Jurkat cells by target cancer cells combined with SynNotch-CAR gated chimeric antigen receptor

[0170] Currently, T cells expressing chimeric antigen receptors (CARs) are effective as a treatment for certain B-cell cancers. However, 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. To achieve this strategy, it is inferred that SynNotch synthetic receptors can be used in the design of therapeutic CAR-T cells, that is, first detecting tumors by binding to tumor-specific cell surface antigens and triggering CAR expression only for a second tumor-specific antigen in the tumor, thereby providing both dual antigen control of CAR-T cell activity and tumor-localized responses.

[0171] Based on this, the inventors designed Jurkat cells combined with SynNotch-CAR gated chimeric antigen receptors (referred to as SynNotch-CAR-Jurkat T cells) containing the corresponding experiments in Jurkat cells (T cell line), specifically including the following steps:

[0172] (1) 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;

[0173] (2) Using conventional methods in the art, construct a single expression of membrane-anchored GFP and a single expression of membrane-anchored CD19 (structure as shown in FIG. Figure 5 As shown, CD19 has the amino acid sequence shown in SEQ ID NO: 12 and the nucleotide sequence shown in SEQ ID NO: 13) and K562 cells co-expressing membrane-anchored GFP and CD19 as transfer cells;

[0174] (3) 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-α 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-α represent the activation of Jurkat T cells.

[0175] like Figure 6 As shown, SynNotch-CAR-Jurkat T cells can be activated only when exposed to K562 expressing two antigens (GFP and CD19).

[0176] MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNW YQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVT CTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSKPTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:10);

[0177]

[0178] METDTLLLWVLLLWVPGSTGDYPYDVPDYAGAQPARPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVDEQKLISEEDLNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR(SEQ ID NO:12);

[0179]

[0180] 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)

[0181] 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 inventors confirmed that it can also play a role in other immune cells by applying the same SynNotch-CAR system as described in Example 4 in NK-92 cells, which specifically includes the following steps:

[0182] (1) 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 packaged with lentivirus 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). The above two viruses are simultaneously infected with NK-92 cells, and 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;

[0183] (2) constructing Huh7 cells expressing membrane-anchored GFP alone, membrane-anchored CD19 alone (for specific sequences, see step (2) of Example 3), and co-expressing membrane-anchored GFP and CD19 as transfer cells;

[0184] (3) labeling the transferred cells described in (2) with CTV dye (purchased from Thermo Fisher);

[0185] (4) The three types of Huh7 cells in step (3) were co-incubated with the NK-92 cells constructed in step (1), and the cells were collected after 48 hours. Dead cells were labeled with PI dye, and the live and dead status of Huh7 cells in each group was analyzed and detected. For details, see Figure 7 Among them, the death ratio of Huh7 cells represents the killing of NK-92 cells on the transmission cells.

[0186] like Figure 7 As shown, 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. Among them, compared with the combination of SynNotch-CAR (M-Syn-CAR) containing SynNotch synthetic receptor 2, 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.

[0187] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0188] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Use of an isolated polypeptide in preparing a SynNotch synthetic receptor, wherein the amino acid sequence of the isolated polypeptide is shown in SEQ ID NO:

1.

2. A SynNotch synthetic receptor, characterized in that include: An extracellular region, wherein the extracellular region has an activity of binding to a first molecule, wherein the first molecule is 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; A transmembrane region, wherein the N-terminus of the transmembrane region is connected to the C-terminus of the extracellular region; An intracellular region, wherein the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region; Wherein, the transmembrane region is an isolated polypeptide, and the amino acid sequence of the isolated polypeptide is shown in SEQ ID NO:

1.

3. The SynNotch synthetic receptor according to claim 2, characterized in that The first molecule is selected from tumor antigens.

4. The SynNotch synthetic receptor according to claim 3, characterized in that The tumor antigen is selected from at least one of a tumor-associated antigen and a tumor-specific antigen.

5. The SynNotch synthetic receptor according to claim 4, characterized in that The tumor antigen is a tumor-specific antigen; 6. The SynNotch synthetic receptor according to claim 2, characterized in that The first molecule is selected from 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, CD156, 8. at least one of 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 and VEGFR-2.

7. The SynNotch synthetic receptor according to claim 2, characterized in that The extracellular region is selected from a first binding protein or a fragment thereof that binds to the first molecule.

8. The SynNotch synthetic receptor according to claim 7, characterized in that The first binding protein or fragment thereof is selected from at least one of an antibody or a functional fragment thereof, a receptor, a ligand of a receptor, and a cell adhesion molecule.

9. The SynNotch synthetic receptor according to claim 7, characterized in that The first binding protein or fragment thereof is a monoclonal antibody or a multispecific antibody that binds to the first molecule.

10. The SynNotch synthetic receptor according to claim 9, characterized in that The monoclonal antibody is selected from at least one of a Fab antibody, a F(ab')2 fragment, a Fv antibody, a single-chain antibody, and a single-domain antibody.

11. The SynNotch synthetic receptor according to claim 2, characterized in that The intracellular region is selected from 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.

12. The SynNotch synthetic receptor according to claim 2, characterized in that The intracellular region is selected from at least one of GaL4-VP64, GaL4-VP16, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, HAP1-VP16, LexA-VP64, Cas9 and Cas13.

13. The SynNotch synthetic receptor according to claim 12, characterized in that The Gal4-VP64 has an amino acid sequence as shown in SEQ ID NO:

2.

14. The SynNotch synthetic receptor according to claim 2, characterized in that The transmembrane region and the intracellular region have the amino acid sequence shown in SEQ ID NO:

3.

15. The SynNotch synthetic receptor according to claim 2, characterized in that The chimeric polypeptide has an amino acid sequence as shown in SEQ ID NO:

4.

16. A first nucleic acid molecule, characterized in that The first nucleic acid molecule encodes the SynNotch synthetic receptor according to any one of claims 2 to 15.

17. The first nucleic acid molecule according to claim 16, characterized in that The first nucleic acid molecule is DNA.

18. A first expression vector, characterized in that: Carrying the first nucleic acid molecule according to any one of claims 16 to 17.

19. The first expression vector according to claim 18, characterized in that: The first expression vector is a eukaryotic expression vector, a prokaryotic expression vector, or a virus.

20. The first expression vector according to claim 18, characterized in that: The first expression vector is a phage or plasmid expression vector.

21. The first expression vector according to claim 18, characterized in that: The first expression vector is a plasmid expression vector.

22. A recombinant cell, characterized in that include: Carrying the first nucleic acid molecule according to any one of claims 16 to 17 or the first expression vector according to any one of claims 18 to 21; or, Expressing the SynNotch synthetic receptor according to any one of claims 2 to 15.

23. The recombinant cell according to claim 22, characterized in that The recombinant cell is obtained by introducing the first expression vector according to any one of claims 18 to 21 into a host cell.

24. The recombinant cell according to claim 23, characterized in that The host cell is selected from at least one of immune cells, neurons, progenitor cells or precursor cells, epithelial cells, endothelial cells and stem cells.

25. The recombinant cell according to claim 23, characterized in that The host cell is selected from at least one of T cells, B cells, monocytes, NK cells, dendritic cells, or macrophages.

26. The recombinant cell according to claim 23, characterized in that The host cell is selected from at least one of a regulatory T cell, a helper T cell, a cytotoxic T cell, a NKT cell and a γδT cell.

27. The recombinant cell according to claim 22, characterized in that The recombinant cell further comprises: A second nucleic acid molecule encoding a chimeric antigen receptor, NK cell receptor or T cell receptor or a second expression vector carrying the second nucleic acid molecule; or expressing the chimeric antigen receptor, NK cell receptor or T cell receptor.

28. The recombinant cell according to claim 22, characterized in that The intracellular region is selected from 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, which is used to bind to the transcriptional activator protein.

29. A pharmaceutical composition, characterized in that comprising the SynNotch synthetic receptor according to any one of claims 2 to 15, the first nucleic acid molecule according to any one of claims 16 to 17, the first expression vector according to any one of claims 18 to 21, or the recombinant cell according to any one of claims 22 to 28; Optionally, a pharmaceutically acceptable excipient is further included.

30. A method for activating immune cells for purposes other than disease diagnosis and treatment, characterized in that: include: contacting the immune cell with a first molecule, wherein the immune cell expresses the SynNotch synthetic receptor of any one of claims 2 to 15; Wherein, the first molecule is a tumor antigen.

31. The method according to claim 30, characterized in that The intracellular region of the chimeric polypeptide is selected from a transcriptional activator protein, the immune cell expresses a chimeric antigen receptor, a NK cell receptor or a T cell receptor, the chimeric antigen receptor, the NK cell receptor or the T cell receptor is selected from an antibody or a functional fragment thereof that binds to a predetermined antigen, and 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 comprises: After the first contact, the immune cell releases the transcriptional activator protein and expresses the chimeric antigen receptor, NK cell receptor or T cell receptor; The chimeric antigen receptor, NK cell receptor or T cell receptor of the immune cell is bound to the predetermined antigen to activate the immune cell.

32. The method according to claim 30, characterized in that The first molecule is selected from 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, CD8 0. CD86, CD94, CD95, CD133, CD134, CD140, CD152, CD154, CD158, CD178, CD181, CD182, CD183, CD200, CD210, CD221, CD246, CD252, CD253, CD261, CD262, CD 273, 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. At least one of PSMA, MAGEA1, 4-1BB, 5T4, BAFF, CA242, CA-IX, MET, CCR4, CNTO888, FAP, MORAb-009, VEGF-A, VEGFR-1 and VEGFR-2.

33. The method according to any one of claims 30 to 32, characterized in that: The immune cells are selected from 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 γδT cells.

34. A method for tracking first cell-second cell contact for non-disease diagnosis and treatment purposes, characterized in that: include: The first cell and the second cell are brought into second contact, wherein the first cell expresses the SynNotch synthetic receptor and the reporter gene protein according to any one of claims 2 to 15, the intracellular region of the chimeric polypeptide is a transcription activator protein, the 5' end of the third nucleic acid molecule encoding the reporter gene 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 gene protein in the first cell, the contact status between the first cell and the second cell is determined.

35. The method according to claim 34, characterized in that The first cell or the second cell is selected from 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.

36. The method according to claim 34 or 35, characterized in that The first cell is selected from 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 γδT cells.

Images