SynNotch receptors and uses thereof

Abstract

The present invention proposes a chimeric polypeptide comprising an extracellular domain having binding activity to a first molecule, a transmembrane domain whose N-terminus is connected to the C-terminus of the extracellular domain, and an intracellular domain whose N-terminus is connected to the C-terminus of the transmembrane domain, wherein the transmembrane domain comprises a separate polypeptide, and the separate polypeptide has at least 80% identity with the transmembrane domain of a Notch receptor protein derived from Xenopus tropicalis.

Description

[Technical Field]

[0001] The field belongs to the field of biotechnology, and specifically, the present invention relates to SynNotch receptors and uses thereof, more specifically, the present invention relates to the use of isolated polypeptides in the preparation of SynNotch synthetic receptors, chimeric polypeptides, first nucleic acid molecules, first expression vectors, recombinant cells, pharmaceutical compositions and uses thereof, methods for activating immune cells, and methods for tracking first cell-second cell contact. [Background technology]

[0002] With the development of synthetic biology, cells can be modified to recognize specific extracellular signals and then transmit the recognized signals to the modified cells, thereby enabling artificially engineered responses and their application in biomedical fields. For example, in addition to the application of chimeric antigen receptor (CAR) technology in clinical treatment, in 2016, Wendell A. Lim and his team disclosed a set of SynNotch receptor systems based on the Notch signaling pathway. These systems can recognize cell membrane surface ligands in the same way, and their signal output is not limited to simple immune cell activation signals but can also achieve various different signal output types, such as the activation and suppression of specific gene expression. This receptor system can achieve various combinations of different input and output signals, has orthogonality between multiple groups of systems within the same system (cell), can operate in many cells, is universal, and has many excellent characteristics such as simplicity and controllability, making it a powerful tool for applying synthetic biology to cell modification.

[0003] In biology, the traditional Notch signaling pathway (system) includes a Notch-dependent ligand, a Notch molecule, and downstream genes regulated by Notch. The Notch molecule is a transmembrane molecule, divided into three parts: an extracellular domain, a transmembrane domain, and an intracellular domain. After the extracellular domain binds to its corresponding Notch ligand, the cell containing the ligand engulfs the Notch molecule, pulling it and exposing the S2 protease cleavage site on the extracellular domain of the Notch molecule, which is then cleaved by members of the ADAM family of metalloproteases. Following cleavage of the extracellular domain, the intracellular domain of the Notch molecule is cleaved by γ-secreta enzyme and released into the cell, where it then enters the nucleus and acts as a transcription factor to regulate the expression of downstream related genes. Wendell A. Lim and his team engineered the mouse Notch1 receptor, replacing its extracellular domain with a single-chain antibody or nanoantibody, and the intracellular domain with a structural domain for transcriptional activation or repression, leaving only the transmembrane structural domain that can be recognized and cleaved by proteases, along with downstream regulatory gene elements. For different targets, the extracellular domain employs a single antibody chain that specifically recognizes the antigen, while the intracellular domain controls the expression of a predetermined target gene or factor. Gene regulatory loops triggered by different antigens can be engineered in the same cell, demonstrating good orthogonality. The SynNotch system can be used in a wide range of cell types, including neurons, tumor cells, epidermal cells, and immune cells. CAR technology and the SynNotch system can be combined to engineer immune cells, achieving "AND-gate" activation, i.e., immune cells can only be activated when two specific surface antigens are simultaneously expressed. By altering the genetic elements downstream of SynNotch, immune cells can secrete therapeutic factors such as single-chain antibodies and cytokines after contacting specific antigens, and these factors exert anti-tumor effects both in vitro and in vivo. This demonstrates that the SynNotch receptor system offers significant technological advantages in cell modification. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art, at least to some extent, by providing a SynNotch receptor, which can enhance the activation ability of downstream genes and reduce background leak activation levels.

[0005] The present invention is completed based on the following findings of the inventors.

[0006] Although the SynNotch system is extremely powerful, the commonly used early version of the SynNotch system (also known as SynNotch synthetic receptor, SynNotch) has the following drawbacks: 1. After activation, the early version of SynNotch does not have a very strong ability to control downstream transcriptional expression. This means that the expression level of downstream genes it activates is not very high. This limits the use of SynNotch, especially when downstream genes require high expression levels for their effectiveness. 2. The early version of SynNotch has a high background leakage level. For genes that require strict downstream control, this high level of leakage expression can lead to the inactivation of the SynNotch molecular switch, causing the molecular expression pathway to directly bypass the SynNotch molecular switch.

[0007] However, through experiments, the inventors unexpectedly discovered that the above problem was due to a lack of a transmembrane domain in the SynNotch molecule. The initial version of the SynNotch synthetic receptor selected the transmembrane domain of the mouse Notch molecule as the transmembrane domain of the SynNotch synthetic receptor. Given the differences in the S2 and S3 cleavage sequences carried by Notch transmembrane domains from different genera and the conformational and sequence preferences of S2 and S3 cleavage enzymes from different species, SynNotch synthetic receptors constructed with different transmembrane domains in cells from different species inevitably have different activation and leakage efficiencies. The inventors' ultimate goal was to engineer human-derived cells with the SynNotch synthetic receptor. During the course of their experiments, they unexpectedly selected the Xenopus Notch transmembrane domain, which increased the transcriptional activation ability and efficiency of the SynNotch synthetic receptor for downstream genes and reduced background leakage activation, allowing the SynNotch synthetic receptor to perform its role more effectively. [Means for solving the problem]

[0008] Therefore, in one aspect, the present invention proposes the use of an isolated polypeptide in the preparation of a SynNotch synthetic receptor, the isolated polypeptide having at least 80% identity with the transmembrane domain of a Notch receptor protein derived from Xenopus tropicalis. The inventors discovered that a SynNotch synthetic receptor prepared by selecting the transmembrane domain of the Notch receptor protein from Xenopus tropicalis can enhance the transcriptional activation ability and efficiency of downstream genes and reduce background leakage of the SynNotch synthetic receptor.

[0009] In another aspect, the present invention provides a chimeric polypeptide. According to an embodiment of the present invention, the chimeric polypeptide comprises an extracellular domain having binding activity to a first molecule, a transmembrane domain whose N-terminus is connected to the C-terminus of the extracellular domain, and an intracellular domain whose N-terminus is connected to the C-terminus of the transmembrane domain, wherein the transmembrane domain comprises a separate polypeptide, and the separate polypeptide has at least 80% identity to the transmembrane domain of a Notch receptor protein derived from Xenopus tropicalis. By selecting the transmembrane domain of the Notch receptor protein of Xenopus tropicalis as the transmembrane domain of the chimeric polypeptide according to the present invention (i.e., a SynNotch synthetic receptor), the chimeric polypeptide according to the present invention can enhance the transcriptional activation ability and efficiency of the chimeric polypeptide to downstream genes and reduce background leakage of the chimeric polypeptide.

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

[0011] In another aspect, the present invention provides a first expression vector. According to an embodiment of the present invention, the first expression vector carries the first nucleic acid molecule described above. After introducing the first expression vector of the present invention into a suitable recipient cell, expression of the chimeric polypeptide described above can be achieved.

[0012] In another aspect, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell comprises the first nucleic acid molecule or the first expression vector described above, or expresses the chimeric polypeptide described above. The recombinant cell of the present invention can effectively express the chimeric polypeptide described above.

[0013] In another aspect, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, or the recombinant cell. As can be seen from the above, cells expressing the chimeric polypeptide can recognize the first molecule and, after binding to the first molecule, can achieve a variety of different signal output types, such as activation and inhibition of specific gene expression. Furthermore, immune cells expressing the chimeric polypeptide can secrete therapeutic factors (e.g., single-chain antibodies, cytokines, etc.) after contact with the first molecule, which can be used for anti-tumor treatment. Furthermore, the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in cells (e.g., immune cells), and the chimeric polypeptide can be expressed in the recombinant cell. Therefore, pharmaceutical compositions comprising the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, or the recombinant cell can be used to target the first molecule and prevent and / or treat related diseases, such as cancer treatment.

[0014] In another aspect, the present invention proposes the use of the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, the recombinant cell or the pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease. As can be seen from the above, cells expressing the chimeric polypeptide can recognize a first molecule and, after binding to the first molecule, can achieve a variety of different signal output types, such as activation and inhibition of expression of a specific gene. Alternatively, immune cells expressing the chimeric polypeptide can be used to secrete therapeutic factors (e.g., single-chain antibodies, cytokines, etc.) after contact with the first molecule, and can be used for anti-tumor purposes. Furthermore, the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in cells (e.g., immune cells), and the chimeric polypeptide can be expressed in the above-mentioned recombinant cells. A pharmaceutical composition can include the above-mentioned chimeric polypeptide, the first nucleic acid molecule, the first expression vector, or the recombinant cells. Thus, a drug comprising the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, the recombinant cells, or the pharmaceutical composition can target the first molecule and be used for the prevention and / or treatment of related diseases such as cancer.

[0015] In another aspect, the present invention provides the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, the recombinant cell, or the pharmaceutical composition described above for use in the prevention and / or treatment of a disease. As can be seen from the above, cells expressing the chimeric polypeptide can recognize a first molecule and, after binding to the first molecule, can achieve a variety of different signal output types, such as activation and repression of specific gene expression. Furthermore, immune cells expressing the chimeric polypeptide can secrete therapeutic factors (e.g., single-chain antibodies, cytokines, etc.) after contact with the first molecule, which can be used for anti-tumor treatment. Furthermore, the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in cells (e.g., immune cells), and the chimeric polypeptide can be expressed in the recombinant cell. Pharmaceutical compositions include the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, or the recombinant cell. Thus, drugs containing the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, the recombinant cell, or the pharmaceutical composition can target the first molecule and be used for the prevention and / or treatment of related diseases, such as cancer treatment.

[0016] In another aspect, the present invention provides a method for treating or preventing a disease. According to an embodiment of the present invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the recombinant cells or pharmaceutical composition described above. As can be seen from the above, cells expressing the chimeric polypeptide can recognize a first molecule and, after binding to the first molecule, achieve a variety of different signal output types, such as activation or repression of specific gene expression. Furthermore, immune cells expressing the chimeric polypeptide can secrete therapeutic factors (e.g., single-chain antibodies, cytokines, etc.) after contact with the first molecule, which can be used for anti-tumor treatment. Furthermore, the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in cells (e.g., immune cells), and the chimeric polypeptide can be expressed in the recombinant cells. A pharmaceutical composition includes the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, or the recombinant cells. Thus, a drug comprising the chimeric polypeptide, the recombinant cells, or the pharmaceutical composition can target the first molecule and be used for the prevention and / or treatment of related diseases, such as cancer.

[0017] In another aspect, the present invention provides a method for activating immune cells. According to an embodiment of the present invention, the method comprises a step of first contacting the immune cells with a first molecule, wherein the immune cells express the chimeric polypeptide described above. Thus, the immune cells expressing the chimeric polypeptide can recognize the first molecule and, after binding to the first molecule, can activate the immune cells.

[0018] In another aspect, the present invention provides a method for tracking contact between a first cell and a second cell. According to an embodiment of the present invention, the method includes a step of bringing the first cell into contact with the second cell, wherein the first cell expresses the chimeric polypeptide and a reporter gene protein described above, the intracellular domain of the chimeric polypeptide is a transcriptional activator protein, the 5' end of a 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 transcriptional activator protein, and the second cell expresses the first molecule. Based on the detection of the reporter gene protein in the first cell, the contact status between the first cell and the second cell is determined. Thus, by employing the above method, the presence or absence of contact between the first cell and the second cell can be determined based on the detection of the reporter gene protein.

[0019] Additional aspects and advantages of the present invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the present invention. [Brief explanation of the drawings]

[0020] The above and / or additional aspects and advantages of the present invention will become apparent and easier to understand from the following description of the embodiments taken in conjunction with the drawings. [Figure 1] FIG. 1 is a schematic diagram showing the structure of a SynNotch synthetic receptor in Example 1 of the present invention. [Figure 2] FIG. 1 is a schematic diagram showing how a SynNotch synthetic receptor cell recognizes a specific extracellular signal in Example 2 of the present invention. [Figure 3] FIG. 1 is a schematic diagram showing the structure of membrane-anchored GFP in Example 2 of the present invention. [Figure 4] 1 shows the leakage efficiency and effective activation efficiency of a SynNotch synthetic receptor containing a mouse Notch transmembrane domain and a SynNotch synthetic receptor containing a Xenopus tropicalis Notch transmembrane domain in Example 2 of the present invention. [Figure 5]FIG. 1 is a schematic diagram showing the structure of membrane-anchored CD19 in Example 3 of the present invention. [Figure 6] 1 shows the results of activation of target cancer cells in response to SynNotch-CAR-Jurkat T cells in Example 3 of the present invention. [Figure 7] This shows the killing efficiency of SynNotch-CAR-NK cells against target cancer cells in Example 4 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0021] The following detailed description of the present invention will be given with reference to the examples. The examples described below are merely illustrative and are used to explain the present invention, but are not intended to limit the present invention.

[0022] It should be noted that the terms "first" and "second" are used for descriptive purposes only and cannot be understood as expressing or implying relative importance or the number of technical features being shown. Thus, a feature defined by "first" or "second" can explicitly or implicitly include at least one of the feature. In the description of the present invention, "plurality" means at least two, e.g., two, three, etc., unless otherwise specified.

[0023] In this specification, the terms "comprise" or "comprises" are non-limiting expressions, i.e., include the content shown by the present invention but do not exclude the content of other embodiments.

[0024] As used herein, the terms "optionally," "optional," or "optional" generally mean that a described event or circumstance may occur, but does not necessarily occur, and the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur.

[0025] As used herein, the terms "identity," "homology," or "similarity," when describing an amino acid sequence or a nucleic acid sequence relative to a reference sequence, refer to conventional methods for determining the percentage of identical amino acids or nucleotides between two amino acid or nucleic acid sequences, as described, for example, in 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 Institute)). Foundation, Washington, DC. Algorithms for comparing sequences and determining sequence identity include the homology comparison 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. 70:173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410).Computer programs utilizing 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., 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.

[0026] As used herein, the term "at least 80% identical" refers to having at least 80% identity to the respective reference sequence, which 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%.

[0027] As used herein, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into an appropriate host and self-replicate, transferring the inserted nucleic acid molecule into and / or between host cells. Expression vectors include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcribing and / or translating DNA or RNA. Expression vectors further include vectors with various of the above functions. The expression vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into an appropriate host cell. Generally, the expression vector can produce a desired expression product by culturing an appropriate host cell containing the expression vector.

[0028] As used herein, the term "recombinant cell" refers to a cell that has a unique trait with stable inheritance, typically by modifying or recombining the genetic material of a host cell using genetic engineering or cell fusion techniques. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformation" or "transfect" as used herein refer to the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of the present invention and used to express and / or secrete the target protein.

[0029] 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 art of pharmacy. All methods include the step of combining the active ingredient with a vector that constitutes one or more accessory ingredients. Typically, the compositions are prepared by uniformly and intimately combining the active chimeric polypeptide or recombinant cells with a liquid vector, a pulverized solid vector, or both.

[0030] As used herein, the term "pharmaceutically acceptable adjuvant" includes any solvent, solid excipient, diluent, or other liquid excipient, suitable for a particular target dosage form. To the extent that a conventional adjuvant is incompatible with the chimeric polypeptide or recombinant cell of the present invention, e.g., produces any adverse biological effect or interacts in a deleterious manner with any other component of the pharmaceutically acceptable composition, it is also contemplated by the present invention.

[0031] As used herein, the term "administration" means introducing a predetermined amount of a substance into a patient in a suitable manner. The recombinant cells or pharmaceutical compositions of the present invention can be administered by any common method as long as they can reach the desired tissue. Various modes of administration are possible, such as peritoneal, intravenous, intramuscular, and subcutaneous injection, but the present invention is not limited to these exemplary modes of administration. Preferably, the compositions of the present invention are administered by intravenous or subcutaneous injection.

[0032] As used herein, the term "treatment" means the use of a drug to obtain a desired pharmacological and / or physiological effect. The effect may be preventative, meaning that a disease or its symptoms are completely or partially prevented, and / or therapeutic, meaning that a disease and / or its adverse effects are partially or completely cured. As used herein, "treatment" covers mammalian, particularly human, diseases and includes (a) preventing the disease or occurrence of a disease in an individual who is susceptible to the disease but has not yet been diagnosed with the disease, (b) suppressing the disease, e.g., preventing the progression of the disease, or (c) alleviating the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treatment" includes any administration of a chimeric polypeptide, recombinant cell, pharmaceutical composition, or drug to an individual to treat, cure, alleviate, ameliorate, mitigate, or prevent a disease in an individual, including, but not limited to, administering a drug comprising a chimeric polypeptide, recombinant cell, or pharmaceutical composition described herein to an individual in need thereof.

[0033] The present invention proposes a use 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 contact between a first cell and a second cell, each of which is described in detail below.

[0034] Use of isolated polypeptides in the preparation of SynNotch synthetic receptors

[0035] In one aspect, the present invention proposes the use of an isolated polypeptide in the preparation of a SynNotch synthetic receptor, the isolated polypeptide having at least 80% identity with the transmembrane domain of a Notch receptor protein derived from Xenopus tropicalis. The inventors discovered that a SynNotch synthetic receptor prepared by selecting the transmembrane domain of the Notch receptor protein from Xenopus tropicalis can enhance the transcriptional activation ability and efficiency of downstream genes and reduce background leakage of the SynNotch synthetic receptor.

[0036] According to an embodiment of the present invention, the isolated polypeptide has 100% identity to the transmembrane domain of the Notch receptor protein from Xenopus tropicalis.

[0037] According to an embodiment of the present invention, the transmembrane domain of the Notch receptor protein from Xenopus tropicalis has the amino acid sequence shown in SEQ ID NO:1. ILDYGFIGGLGKNITPDNEEICENEQCAELADNKICNANNINHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFNDGKCDSQCNNSGCLYDGFDCQKVEVQCNPLYDQYCRDHFQDGHCDQGCNNAECEWDGLDCDNMPENLAEGTLLIVVLMPPEKLKNNSVNFLRE LSRVLHTNVVFKKDSKGEYKIYPYYGNEEELKKHHIKKRSAASWSDAPTAIFSTMKESVLPGRRRRELDQMEVRGSIVYLEIDNRQCYKSSSQCFTSATDVAAFLGALATHGNLNIPYKIEAVKSEIVETAKPPPPLYAMFSMLVIPLLIIFVIMVVIVNKKRRR(SEQ ID NO:1).

[0038] Chimeric Polypeptides

[0039] In another aspect, the present invention provides a chimeric polypeptide. According to an embodiment of the present invention, the chimeric polypeptide comprises an extracellular domain having binding activity to a first molecule, a transmembrane domain whose N-terminus is connected to the C-terminus of the extracellular domain, and an intracellular domain whose N-terminus is connected to the C-terminus of the transmembrane domain, wherein the transmembrane domain comprises a separate polypeptide, and the separate polypeptide has at least 80% identity to the transmembrane domain of a Notch receptor protein derived from Xenopus tropicalis.

[0040] The inventors discovered that by selecting the transmembrane region of the Xenopus tropicalis Notch receptor protein as the transmembrane region of a chimeric polypeptide (i.e., a SynNotch synthetic receptor), the chimeric polypeptide can enhance the transcriptional activation ability and efficiency of downstream genes and reduce background leakage of the chimeric polypeptide. Furthermore, cells expressing the chimeric polypeptide can recognize a first molecule and, after binding to the first molecule, achieve a variety of different signal output types, such as activation and repression of specific gene expression, or can be used to prepare immune cells expressing the chimeric polypeptide. After contact with the first molecule, the immune cells can secrete factors with therapeutic effects (e.g., single-chain antibodies, cytokines, etc.) for anti-tumor use.

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

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

[0043] According to an embodiment of the present invention, the transmembrane domain further comprises an epidermal growth factor-like repeat (EGF repeat) and / or a RAM sequence, which can further reduce background leak activation of the SynNotch synthetic receptor.

[0044] 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. VVSPCASRPCYNGGTCQFSPEEPFFQCFCPTNFNGLFCH(SEQ ID NO:15).

[0045] According to an embodiment of the present invention, the RAM sequence is the amino acid sequence as shown in SEQ ID NO:16. EHGQLWFP (SEQ ID NO: 16).

[0046] According to an embodiment of the present invention, the C-terminus of the EGF-like repeat sequence is connected to the N-terminus of the separated polypeptide, and / or the C-terminus of the separated polypeptide is connected to the N-terminus of the RAM sequence.

[0047] According to an embodiment of the present invention, the first molecule comprises 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.

[0048] As used herein, the term "tumor antigen" generally refers to an antigenic substance that emerges or is overexpressed during the development and progression of a tumor. Tumor antigens are classified into tumor-specific antigens and tumor-associated antigens. Tumor-specific antigens (TSAs) are novel antigens that are specific to tumor cells or are present only in certain tumor cells and not in normal cells. Tumor-associated antigens (TAAs) are antigens that are specific to non-tumor cells and are also present in normal cells and other tissues, and their abundance significantly increases during cell carcinogenesis. These antigens include, but are not limited to, PD-L1, PD-1, TGF-β, CEA, GD2, and GD3.

[0049] As used herein, the term "cellular receptor" or "receptor" should be understood broadly and refer to a molecule that is located on the cell membrane and can recognize and bind to various extracellular signal molecules (ligands), including, but not limited to, growth factor receptors (e.g., VEGF receptors), (NKG2D polypeptides (receptors for MICA, MICB, and ULB6), cytokine receptors (e.g., IL-13 receptor, IL-2 receptor, etc.), epidermal growth factor (EGF) receptor, Her2, CD27, natural cytotoxicity receptors (NCRs) (e.g., NKP30 (NCR3 / CD337) polypeptides (receptors for HLA-B-associated transcript 3 (BAT3) and B7-H6)), T cell antigen receptors, dihydrofolate receptors, chimeric cytokine receptors, Fc receptors, extracellular matrix receptors (e.g., integrins), cell adhesion receptors (e.g., cadherins), immunoregulatory receptors (positive co-receptors (e.g., CD28) and negative (immunosuppressive) co-receptors (e.g., PD1)), and receptors for immunoregulatory molecules (e.g., TGFβ).

[0050] As used herein, the term "ligand for a cell receptor" should be understood broadly and refers to a chemical substance that can bind to and interact with a cell membrane receptor to produce a specific biological effect, such as, for example, 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 containing the sequence Arg-Gly-Asp), N-polysaccharides, etc.

[0051] Illustratively, the ligand is VEGF and the receptor is a VEGF receptor, or the ligand is heregulin and the receptor is Her2.

[0052] As used herein, the term "cytokine" should be understood broadly to refer to proteins or small polypeptides that transmit information between cells and have immunoregulatory and effector functions, such as IL-10. The term "cytokine receptor" should be understood broadly to refer to receptors that can bind to cytokines on the surface of cells, such as Her2 and IL-10R.

[0053] In one preferred embodiment of the invention, the tumor antigen is a tumor-specific antigen.

[0054] According to an embodiment of the present invention, the first molecule is 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, CD 158, 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.

[0055] According to an embodiment of the present invention, the extracellular domain comprises a first binding protein or a fragment thereof that binds to the first molecule; According to an embodiment of the present invention, the first binding protein or fragment thereof comprises at least one of an antibody or functional fragment thereof, a receptor, a ligand of a receptor, and a cell adhesion molecule.

[0056] As used herein, "cell adhesion molecule" or "CAM" can refer to a component or polypeptide of a binding cell surface molecule that binds to the extracellular matrix (ECM). For example, the cell adhesion molecule can be the extracellular region of a CAM; the CAM can be a calcium-independent adhesion molecule, such as an immunoglobulin superfamily CAM; the CAM can also be a calcium-dependent adhesion molecule, such as an integrin, cadherin, or selectin; the cell adhesion molecule can be an integrin, such as a cadherin, e.g., E-cadherin, P-cadherin, N-cadherin, R-cadherin, or M-cadherin; the cell adhesion molecule can be a selectin, e.g., E-selectin, L-selectin, or P-selectin.

[0057] As used herein, the term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, but the specific structure is not limited as long as they exhibit the required biological activity. Antibody molecules typically comprise a light chain with a low molecular weight and a heavy chain with a high molecular weight, with the heavy chain (H chain) and light chain (L chain) connected by disulfide bonds. The amino acid sequence at the amino terminal (N-terminal) of the peptide chain varies greatly and is called the variable region (V region), while the carboxyl terminal (C-terminal) is relatively stable and changes little and is called the constant region (C region). The V regions of the L chain and H chain are called VL and VH, respectively.

[0058] As used herein, the terms "full length antibody," "full length single antibody," or "full length monoclonal antibody" all comprise 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).

[0059] As used herein, the term "functional fragment" refers to a fragment containing part or all of an antibody, lacking at least some of the amino acids present in the full-length chain, but retaining the functional activity of being able to specifically bind to an antigen; for example, the fragment contains part or all of the antibody CDRs. Such fragments retain biological activity, bind to an antigen, and compete with other antigen-binding molecules (including complete antibodies) for binding to a predetermined epitope. Such fragments are selected from Fab, Fv, scFv, or single-domain antibodies. Such fragments may be produced by recombinant nucleic acid technology or by enzymatic or chemical degradation of antigen-binding molecules (including complete antibodies).

[0060] According to an embodiment of the present invention, the first binding protein or fragment thereof is a monoclonal or polyclonal antibody that binds to the first molecule.

[0061] As used herein, the term "polyclonal antibody" has the same meaning as "multispecific antibody," and refers to an antibody capable of recognizing multiple antigen epitopes, such as an antibody capable of recognizing two antigen epitopes (bispecific antibody, abbreviated as double antibody), three antigen epitopes, or four antigen epitopes. This should be understood in a broad sense, and the specific structure is not limited, as long as it can recognize multiple antigen epitopes. In the present invention, at least one of the multiple antigen epitopes is derived from the first molecule.

[0062] According to an embodiment of the present invention, the monoclonal antibody comprises at least one of a Fab antibody, a F(ab')2 fragment, an Fv antibody, a single chain antibody, a single domain antibody, and a minimal recognition unit.

[0063] As used herein, the terms "single domain antibody," "nanobody," and "VHH antibody" can be used interchangeably and refer to antigen-binding immunoglobulin (variable) domains originally described as "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)), which contain a heavy chain variable region (VH) and conventional CH2 and CH3 regions, and which specifically bind to an antigen protein (e.g., D-dimer) via the heavy chain variable region.

[0064] As used herein, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only the Fab molecule, which consists of the VH and CH1 of a heavy chain and an intact light chain, connected by a single disulfide bond between the light and heavy chains.

[0065] As used herein, the term "F(ab')2 antibody" or "F(ab')2 fragment" comprises two antigen-binding F(ab') portions connected by disulfide bonds.

[0066] As used herein, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment in which only the light chain variable region (VL) and heavy chain variable region (VH) are non-covalently connected, and is the smallest functional fragment of an antibody molecule in which an intact antigen-binding site remains.

[0067] As used herein, the terms "single-chain antibody" and "scFv fragment" refer to an antibody or fragment in which the antibody heavy chain variable region and light chain variable region are connected by a short peptide.

[0068] As used herein, the terms "minimal recognition unit" and "MRU" refer to an antibody or fragment consisting of only one CDR, and its molecular weight is sufficiently small that it accounts for only about 1% of the entire antibody.

[0069] According to an embodiment of the present invention, the intracellular domain comprises at least one of a transcriptional activator protein, a transcriptional inhibitory protein, a transcription factor, a site-specific nuclease, a recombinase, an activating immunoreceptor intracellular structural domain, and an inhibitory immunoreceptor intracellular structural domain.

[0070] According to an embodiment of the present invention, the intracellular domain comprises at least one of GaL4-VP64, GaL4-VP16, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, HAP1-VP16, LexA-VP64, Cas9, and Cas13.

[0071] According to an embodiment of the present invention, the Gal4-VP64 has an amino acid sequence as shown in SEQ ID NO:2. MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSAAAGGSGGSGGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFLDMLGS(SEQ ID NO:2).

[0072] According to an embodiment of the present invention, the transmembrane and intracellular domains have the amino acid sequence as set forth in SEQ ID NO:3. (SEQ ID NO:3).

[0073] According to an embodiment of the invention, the chimeric polypeptide has an amino acid sequence as shown in SEQ ID NO:4. (SEQ ID NO:4).

[0074] Nucleic acid molecules, vectors, recombinant cells and pharmaceutical compositions

[0075] In another aspect, the present invention provides a first nucleic acid molecule. According to an embodiment of the present invention, the first nucleic acid molecule encodes a claimed chimeric polypeptide. The chimeric polypeptide can be obtained by encoding the first nucleic acid molecule according to an embodiment of the present invention.

[0076] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0077] It should be understood by those skilled in the art that the first nucleic acid molecule referred to herein actually includes either one or both of the complementary strands. For convenience, only one strand is often shown herein, but the complementary strand is also disclosed. Furthermore, the molecular sequences of the present invention include DNA and RNA formats, and the disclosure of one strand implies the disclosure of the other.

[0078] In another aspect, the present invention provides a first expression vector. According to an embodiment of the present invention, the first expression vector comprises the first nucleic acid molecule described above. When the first nucleic acid molecule is connected to the vector, the first nucleic acid molecule can be directly or indirectly connected to control elements on the vector, and these control elements may control the translation and expression of the first nucleic acid molecule. Of course, these control elements may be derived directly from the vector itself or may be exogenous, i.e., not derived from the vector itself. Of course, the first nucleic acid molecule may be operably connected to the control elements.

[0079] As used herein, "operably linked" refers to connecting a foreign gene to a vector so that control elements within the vector, such as transcriptional and translational control sequences, can function to regulate the transcription and translation of the desired foreign gene. Commonly used vectors include plasmids and bacteriophages. In some specific embodiments of the present invention, after introducing the vector into an appropriate recipient cell (also referred to as a recipient cell or host cell), expression of the chimeric polypeptide described above can be effectively achieved via a regulatory system.

[0080] 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.

[0081] According to an embodiment of the present invention, the first expression vector is a plasmid expression vector.

[0082] In another aspect, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell comprises the first nucleic acid molecule or the first expression vector described above, or expresses the chimeric polypeptide described above. The recombinant cell can be used to effectively express the chimeric polypeptide described above in the recombinant cell under appropriate conditions.

[0083] The "appropriate conditions" referred to in the present invention refer to conditions suitable for the expression of the chimeric polypeptide described above. Those skilled in the art can easily understand that the appropriate conditions for the expression of the chimeric polypeptide described above include, but are not limited to, an appropriate transformation or transfection method, appropriate transformation or transfection conditions, a healthy cell state, an appropriate cell density, an appropriate cell culture environment, and an appropriate cell culture time. The "appropriate conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the chimeric polypeptide according to the specific environment of the laboratory.

[0084] According to an embodiment of the present invention, the recombinant cell is obtained by introducing the first expression vector described above into a host cell.

[0085] According to an embodiment of the present invention, the host cells include at least one of immune cells, neurons, progenitor or precursor cells, epithelial cells, endothelial cells, and stem cells.

[0086] According to an embodiment of the present invention, the host 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.

[0087] According to an embodiment of the present invention, the recombinant cell 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 the chimeric antigen receptor, T cell receptor, or NK cell receptor has been discovered. Experiments by the inventors have shown that the chimeric polypeptide of the present invention can be used in combination with a chimeric antigen receptor, an NK cell receptor, or a T cell receptor to jointly modify immune cells (e.g., Jurkat cells and NK cells), and the immune cells bearing the chimeric polypeptide + CAR / TCR combination can recognize the corresponding ligand, efficiently activating the immune cells (e.g., Jurkat T cells and NK cells) and killing targets (e.g., tumor cells).

[0088] As used herein, the term "chimeric antigen receptor (CAR)" refers to a fusion protein comprising an extracellular structural domain capable of binding to an antigen, a transmembrane structural domain derived from a polypeptide different from the extracellular structural domain, and at least one intracellular structural domain. A "chimeric antigen receptor (CAR)" is also referred to as a "chimeric receptor," "T-body," or "chimeric immune receptor (CIR)." The "extracellular structural domain capable of binding to an antigen" refers to any oligopeptide or polypeptide capable of binding to a certain antigen. The "intracellular structural domain" refers to any known oligopeptide or polypeptide that functions as a structural domain that transmits a signal to activate or inhibit a biological process within a cell.

[0089] As used herein, the term "T cell receptor (abbreviated as TCR)" refers to a molecule found on the surface of T cells that recognizes antigens that can bind to MHC molecules. Naturally occurring TCR isodimers consist of alpha (α) and beta (β) chains in approximately 95% of T cells, while approximately 5% of T cells have TCRs consisting of gamma (γ) and delta (δ) chains. During antigen processing, antigens are degraded intracellularly and then transported to the cell surface by major histocompatibility complex (MHC) molecules. T cells can recognize this antigen peptide-MHC complex on the surface of antigen-presenting cells. Binding of the TCR to the antigen peptide-MHC complex results in activation of the T lymphocyte, which expresses the TCR through a series of biochemical reactions mediated by associated enzymes, co-receptors, and specialized accessory molecules on the T lymphocyte. The MHC molecule may be a class I or class II MHC molecule. The complex is found on antigen-presenting cells, such as dendritic cells, B cells, or any other cells (e.g., K562 cells). The human leukocyte antigen system (HLA) is the name for 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 primarily intracellular proteins, i.e., peptides derived from proteins expressed within cells.

[0090] As used herein, the term "NK cell receptor" refers to a molecule found on the surface of NK cells. It can be divided into inhibitory receptors and activating receptors depending on the function they mediate. Inhibitory receptors recognize specific molecules expressed on the surface of normal cells, then transmit a killing inhibitory signal to suppress the cell's 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 recognize corresponding ligands on the surface of target cells, then transmit an activating signal into the cell to exert killing effects. Activating receptors include, but are not limited to, NKp30, NKp44, NKp46, and CD16.

[0091] According to an embodiment of the present invention, the intracellular domain comprises 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 nucleic acid sequence, which is used to bind to the transcriptional activator protein.

[0092] Illustratively, the intracellular domain is GaL4-VP64, and the inducible expression nucleic acid sequence is a UAS-minimal-CMV sequence.

[0093] The UAS-minimal-CMV sequence has the nucleic acid sequence as shown in SEQ ID NO:14. GGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGGAGCATGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGA CTAGTTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGC(SEQ ID NO:14).

[0094] In a preferred 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, a chimeric antigen receptor binds a second molecule primarily via its extracellular domain.

[0095] According to an embodiment of the present invention, the second molecule comprises 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.

[0096] In one preferred embodiment of the invention, the second molecule is selected from the group consisting of 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, CD and at least one of D158, 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.

[0097] In another aspect, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, or the recombinant cell. As can be seen from the above, cells expressing the chimeric polypeptide can recognize the first molecule and, after binding to the first molecule, can achieve a variety of different signal output types, such as activation and inhibition of specific gene expression. Furthermore, immune cells expressing the chimeric polypeptide can secrete therapeutic factors (e.g., single-chain antibodies, cytokines, etc.) after contact with the first molecule, which can be used for anti-tumor treatment. Furthermore, the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in cells (e.g., immune cells), and the chimeric polypeptide can be expressed in the recombinant cell. Therefore, pharmaceutical compositions comprising the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, or the recombinant cell can be used to target the first molecule and prevent and / or treat related diseases, such as cancer treatment.

[0098] According to an embodiment of the present invention, the composition further comprises a pharmaceutically acceptable adjuvant.

[0099] use

[0100] In another aspect, the present invention proposes the use of the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, the recombinant cell or the pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease. As can be seen from the above, cells expressing the chimeric polypeptide can recognize a first molecule and, after binding to the first molecule, can achieve a variety of different signal output types, such as activation and inhibition of expression of a specific gene. Alternatively, immune cells expressing the chimeric polypeptide can be used to secrete therapeutic factors (e.g., single-chain antibodies, cytokines, etc.) after contact with the first molecule, and can be used for anti-tumor purposes. Furthermore, the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in cells (e.g., immune cells), and the chimeric polypeptide can be expressed in the above-mentioned recombinant cells. A pharmaceutical composition can include the above-mentioned chimeric polypeptide, the first nucleic acid molecule, the first expression vector, or the recombinant cells. Thus, a drug comprising the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, the recombinant cells, or the pharmaceutical composition can target the first molecule and be used for the prevention and / or treatment of related diseases such as cancer.

[0101] In another aspect, the present invention provides the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, the recombinant cell, or the pharmaceutical composition for preventing and / or treating a disease. As can be seen from the above, cells expressing the chimeric polypeptide can recognize a first molecule and, after binding to the first molecule, achieve a variety of different signal output types, such as activation and repression of specific gene expression. Furthermore, immune cells expressing the chimeric polypeptide can secrete therapeutic factors (e.g., single-chain antibodies, cytokines, etc.) after contact with the first molecule, which can be used for anti-tumor treatment. Furthermore, the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in cells (e.g., immune cells), and the chimeric polypeptide can be expressed in the recombinant cell. Pharmaceutical compositions include the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, or the recombinant cell. Thus, drugs containing the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, the recombinant cell, or the pharmaceutical composition can target the first molecule and be used for preventing and / or treating related diseases, such as cancer treatment.

[0102] According to an embodiment of the present invention, the use of the above two aspects includes at least one of the following technical features:

[0103] According to an embodiment of the present invention, the disease includes cancer or tumor, and immune-related disease.

[0104] According to an embodiment of the present invention, the diseases include cancer or tumor, autoimmune disease, inflammation and diseases related to cellular senescence.

[0105] As used herein, the term "cancer" or "tumor" may refer to uncontrolled cell growth, such as non-small cell lung cancer, papillary thyroid carcinoma, pleomorphic glioma, 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, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, cervical cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma, or gastric cancer.

[0106] method

[0107] In another aspect, the present invention provides a method for activating immune cells. According to one embodiment of the present invention, the method includes a first step of contacting the immune cells with a first molecule, wherein the immune cells express the chimeric polypeptide described above. Thus, the immune cells expressing the chimeric polypeptide can recognize the first molecule and activate the immune cells after binding to the first molecule. This method is particularly applicable to scientific research, where the activated immune cells are obtained by in vitro cell culture of the immune cells.

[0108] According to an embodiment of the present invention, the intracellular domain of the chimeric polypeptide comprises 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 comprises an antibody or a functional fragment thereof that binds to a predetermined antigen; the 5' end of the second nucleic acid molecule encoding the chimeric antigen receptor, the NK cell receptor, or the T cell receptor is connected to an inducible expression nucleic acid sequence, which is used to bind to the transcriptional activator protein; and the method further comprises, after the first contact, the immune cell releasing the transcriptional activator protein and expressing the chimeric antigen receptor, the NK cell receptor, or the T cell receptor; and binding the chimeric antigen receptor, the NK cell receptor, or the T cell receptor of the immune cell to a predetermined antigen, thereby activating the immune cell.

[0109] According to an embodiment of the present invention, the inducible nucleic acid sequence has the nucleic acid sequence as shown in SEQ ID NO:14.

[0110] According to an embodiment of the present invention, the first molecule is a tumor antigen.

[0111] In one preferred embodiment of the invention, the first molecule is selected from the group consisting of 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, CD and at least one of D158, 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.

[0112] 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.

[0113] In another aspect, the present invention provides a method for tracking contact between a first cell and a second cell. According to one embodiment of the present invention, the method includes a step of bringing the first cell into contact with the second cell, wherein the first cell expresses the chimeric polypeptide and a reporter gene protein described above, the intracellular domain of the chimeric polypeptide is a transcriptional activator protein, the 5' end of a third nucleic acid molecule encoding the reporter gene protein is connected to an inducible expression nucleic acid sequence, the inducible expression nucleic acid sequence being used to bind to the transcriptional activator protein, and the second cell expresses the first molecule. The contact between the first cell and the second cell is determined based on the detection of the reporter gene protein in the first cell. This method can thus determine whether or not contact between the first cell and the second cell has occurred based on the detection of the reporter gene protein, making it particularly applicable to scientific research, where the method can be used to determine whether or not contact between two types of cells has occurred in vitro.

[0114] According to an embodiment of the present invention, the inducible nucleic acid sequence has the nucleic acid sequence as shown in SEQ ID NO:14.

[0115] According to embodiments of the present invention, the first molecule may be located on the surface of a second cell, immobilized on an insoluble substrate, present in an extracellular matrix, present in an artificial matrix, or may be soluble.

[0116] According to an embodiment of the present invention, the first molecule is a tumor antigen.

[0117] In one preferred embodiment of the invention, the first molecule is selected from the group consisting of 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, CD and at least one of D158, 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.

[0118] According to an embodiment of the present invention, the first cell or the second cell comprises at least one of an immune cell, a neuron, a progenitor or precursor cell, an epithelial cell, an endothelial cell, and a stem cell.

[0119] According to an embodiment of the present invention, the first 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.

[0120] In another aspect, the present invention provides a method for controlling cellular activity, according to an embodiment of the present invention, the method comprising a third step of contacting the cell with a first molecule, wherein the cell expresses the chimeric polypeptide as described above.

[0121] According to an embodiment of the present invention, the cells include at least one of immune cells, neurons, progenitor or precursor cells, epithelial cells, endothelial cells, and stem cells.

[0122] 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.

[0123] In another aspect, the present invention provides a method for promoting intracellular gene or protein expression. According to an embodiment of the present invention, the method comprises a fourth step of contacting the cell with a first molecule, wherein the cell expresses the chimeric polypeptide described above, the cell harbors the gene or expresses the protein, and the gene comprises a nucleic acid molecule encoding at least one of a chimeric antigen receptor, a second chimeric polypeptide, a translational 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 translational regulator, a cytokine, a hormone, a chemokine, an antibody, and a protein located in the intracellular region.

[0124] According to an embodiment of the invention, the intracellular domain of the chimeric polypeptide is a transcriptional activator protein, and the chimeric antigen receptor, second chimeric polypeptide, translational regulator, cytokine, hormone, chemokine, or antibody comprises a second binding protein of the transcriptional activator protein or a fragment thereof.

[0125] According to an embodiment of the present invention, the cells include at least one of immune cells, neurons, progenitor or precursor cells, epithelial cells, endothelial cells, and stem cells.

[0126] 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.

[0127] In another aspect, the present invention provides a method for treating or preventing a disease. According to an embodiment of the present invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the recombinant cells or pharmaceutical composition described above. As can be seen from the above, cells expressing the chimeric polypeptide can recognize a first molecule and, after binding to the first molecule, achieve a variety of different signal output types, such as activation or repression of specific gene expression. Furthermore, immune cells expressing the chimeric polypeptide can secrete therapeutic factors (e.g., single-chain antibodies, cytokines, etc.) after contact with the first molecule, which can be used for anti-tumor treatment. Furthermore, the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in cells (e.g., immune cells), and the chimeric polypeptide can be expressed in the recombinant cells. A pharmaceutical composition includes the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, or the recombinant cells. Thus, a drug comprising the chimeric polypeptide, the recombinant cells, or the pharmaceutical composition can target the first molecule and be used for the prevention and / or treatment of related diseases, such as cancer.

[0128] The effective amount of the recombinant protein or pharmaceutical composition described in the present invention may vary depending on the mode of administration and the severity of the disease being treated. The selection of a preferred effective amount can be determined by one skilled in the art based on various factors (e.g., clinical trials). These factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life, the severity of the disease being treated in the patient, the patient's body weight, the patient's immune status, and the route of administration. For example, depending on the exigencies of the therapeutic situation, the protein or pharmaceutical composition may be administered in divided doses per day or the dosage may be proportionally reduced.

[0129] The recombinant protein or pharmaceutical composition of the present invention can be formulated into a medicament suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular). These medicaments can be prepared in a variety of formats, such as liquid, semi-solid, and solid dosage forms, including, but not limited to, liquid solutions (e.g., injection and infusion solutions) or lyophilized powders. Typical medicaments are in the form of injection or infusion solutions. The recombinant protein or pharmaceutical composition can be administered by intravenous infusion or injection, or intramuscular or subcutaneous injection.

[0130] According to an embodiment of the present invention, the administration route of the method is subcutaneous injection or intravenous injection.

[0131] 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 cellular senescence-related disease.

[0132] 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 carcinoma, pleomorphic glioma, 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, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, cervical cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma or gastric cancer.

[0133] The following examples are used in combination to illustrate the present disclosure. Those skilled in the art can understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. If no specific techniques or conditions are shown in the examples, they should be carried out according to the techniques or conditions described in the literature of the field or according to the product specifications. If no manufacturer is shown for the reagents or equipment used, they are conventional products that can be purchased commercially.

[0134] Example 1: Design of SynNotch synthetic receptor and construction of its expression plasmid vector

[0135] 1. The structure of a SynNotch synthetic receptor (abbreviated as 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, abbreviated as the transmembrane region), and an intracellular domain (also referred to as the intracellular region). The extracellular domain may be replaced with a single-chain antibody or nanobody, etc., and the intracellular domain may be replaced with a transcriptional activation or repression 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 Xenopus tropicalis Notch transmembrane domain that can be recognized and cleaved by a protease to obtain a modified SynNotch receptor. The structure of a SynNotch receptor modified according to the design of the present invention is shown, for example, in Figure 1. Figure 1 shows the structure of a SynNotch receptor containing a mouse or Xenopus tropicalis Notch transmembrane domain (Notch core).

[0136] 2. Construct an expression plasmid vector for the above SynNotch synthetic receptor. The specific steps are as follows: (1) A SynNotch synthetic receptor containing a Xenopus Notch transmembrane domain (abbreviated 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 a SynNotch synthetic receptor containing a mouse Notch transmembrane domain (abbreviated 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. Then, the nucleotide sequence shown in SEQ ID NO:5 and the nucleotide sequence shown in SEQ ID NO:7 were synthesized in a Puc57 vector by Kim Wei-Ji Co., Ltd. (2) The pCDH-EV vector is double-digested using EocR1 and BamH1 endonucleases, electrophoresed on a 1% agarose gel for 20 minutes, and then the double-digested vector is recovered by rubber cutting. (3) Using the primer pairs, the two pUC57 vectors from step (1) are subjected to PCR to obtain the nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:7, respectively. The nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:7 are then homologously recombined with the double-digested vector obtained in step (2) and the nucleotide fragment of the SynNotch synthetic receptor to obtain two expression plasmid vectors, pCDH-antiGFP-XENTR-SynNotch-Gal4-VP64 and pCDH-antiGFP-MOUSE-SynNotch-Gal4-VP64. (4) The two expression plasmid vectors constructed above (PCDH-antiGFP-XENTR-SynNotch-Gal4VP64 and pCDH-antiGFP-MOUSE-SynNotch-Gal4-VP64) were sequenced, and the sequencing results confirmed that the construction was successful. MALPVTALLLPLALLLHAARPEQKLISEEDLMADVQLVESGGGLVQAGGSLRLSCAASGRTISMAAMSWFRQAPGKEREFVAGISRSAGSAVHADSVKGRFTISRDNTKNTLYLQMNSLKAEDTAVYYCAVRTSGFFGSIPRTGTAFDYWGQGTQVTVSILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRRMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSAAAGGSGGSGGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGS(SEQ ID NO:6);

[0137] Example 2: Cellular testing and validation of SynNotch receptors

[0138] In this example, the specific process by which SynNotch receptor cells recognize a specific extracellular signal is as follows: when the anti-GFP antibody in the extracellular domain of the SynNotch receptor binds in a single chain to the GFP antigen in the sender cell, the Notch transmembrane domain is cleaved by a protease, releasing GAL4VP64 into the cell, which then specifically recognizes and binds to the GAL4 binding site (UAS region). The VP64 transcriptional activation element then activates the transcription of the downstream BFP gene, promoting the expression of the BFP fluorescent protein. See Figure 2 for details.

[0139] In the present invention, we used HT1080 cells as sender cells and another lot of HT1080 cells as receiver cells to verify at the cellular level whether the expression plasmid vector (pCDH-antiGFP-XENTR-SynNotch-Gal4VP64) prepared in Example 1 enhances the transcriptional activation ability of downstream genes. The specific verification steps are as follows: (1) The HT1080 cell line can be infected with slow viruses and stably express membrane-anchored GFP, resulting in transduced cells (the structure of membrane-anchored GFP is shown, for example, in Figure 3, and GFP has the amino acid sequence shown in SEQ ID NO:8 and the nucleotide sequence shown in SEQ ID NO:9).

[0140] The amino acid and nucleotide sequences of GFP are as follows: METDTLLLWVLLLWVPGSTGDMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR(SEQ ID NO:8); ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCCGGCAGCACCGGCGACATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGAACGCCGTGGGCCAGGACACCCAGGAGGTGATCGTGGTGCCCCACAGCCTGCCCTTCAAGGTGGTGGTGATCAGCGCCATCCTGGCCCTGGTGGTGCTGACCATCATCAGCCTGATCATCCTGATCATGCTGTGGCAGAAGAAGCCCCGC(SEQ ID NO:9)。

[0141] Another batch of HT1080 cells was used as recipient cells and first infected with the pHR_Gal4UAS_tBFP_PGK_mCherry slow virus (purchased from Addgene, #79130; after infecting host cells, the slow virus can express the BFP fluorescent protein in response to GAL4VP64). Cells that stably express high levels of pHR_Gal4UAS_tBFP_PGK_mCherry were selected and then infected with the pCDH-antiGFP-XENTR-SynNotch-Gal4VP64 slow virus. pHR_Gal4UAS_tBFP_PGK_mCherry and pCDH-antiGFP-XENTR-SynNotch-Gal4VP64 slow viruses were prepared by co-transfecting the expression plasmids (pCDH-antiGFP-XENTR-SynNotch-Gal4VP64 or pHR_Gal4UAS_tBFP_PGK_mCherry), pMD2.G, and psPAX2 into 15 cm culture dishes containing HEK293-T cells at 90% confluency. After 3 days, the supernatant was collected and concentrated by filtering the virus solution through a 0.45 μM filter. (2) Harvest the above stably transfected recipient cells, HT1080 cells, and culture them at a cell density of 3 × 10 5 The cells were inoculated into a 24-well plate at 3 × 10 cells / mL. 5 HT1080 cells, which can express GFP, were added at 100 cells / mL and co-cultured for 48 hours. Cell images were taken using an Olympus inverted fluorescence microscope, and the fluorescence intensity expressed in the cells was analyzed using ImageJ. The results are shown in Figure 4.

[0142] Figure 4A shows a comparison of fluorescence micrographs. MOUSE-Syn represents the original system, a SynNotch synthetic receptor containing the mouse Notch transmembrane domain (abbreviated as SynNotch synthetic receptor 2), and XENTR-Syn represents the Xenopus tropicalis Notch transmembrane domain (abbreviated as SynNotch synthetic receptor 1). As can be seen in Figure 4A, SynNotch synthetic receptor 1 significantly improves activation efficiency compared to SynNotch synthetic receptor 2.

[0143] Figure 4B shows the mean fluorescence intensity of BFP blue fluorescent protein expressed in HT1080 cells analyzed using ImageJ software. As can be seen from Figure 4B, the activation efficiency of SynNotch synthetic receptor 1 (i.e., XENTR group) was 95.1% higher and the leakage level of SynNotch synthetic receptor 1 (i.e., XENTR group) was 56.4% lower than that of SynNotch synthetic receptor 2 (i.e., MOUSE group).

[0144] There are three types of cell-cell contact: contact between adherent cells, contact between adherent cells and suspension cells, and contact between suspension cells. Because these three contact types reflect the three real contact environments of cells in vivo to some extent, it is important to verify that all three cell contact methods can effectively activate the SynNotch system. Because HT1080 is an adherent cell, the results in Figures 4A and 4B above only demonstrate that adherent cell-to-adherent cell contact can activate the SynNotch system, but not the other two contact systems. Therefore, we further constructed suspension recipient cells, Jurkat, and suspension release cells, K562, using the method described in step (1) of this example. Furthermore, in step (2) of this example, K562 emitter cells were co-cultured with Jurkat recipient cells for 48 hours to observe whether contact between suspension cells and suspension cells could activate the SynNotch system. Concurrently, HT1080 emitter cells were co-cultured with Jurkat recipient cells for 48 hours to observe whether contact between suspension cells and appositional cells could activate the SynNotch system. The results, shown in Figures 4C and 4D, demonstrate that both XENTR-SynNotch and MOUSE-SynNotch were effectively activated by contact between suspension receptor cells and suspension-suspended emitter cells, as well as between suspension-suspended acceptor cells and appositional emitter cells. Furthermore, the leak activation efficiency of XENTR-SynNotch was much lower than that of MOUSE-SynNotch, while the effective activation efficiency was slightly higher than that of MOUSE-SynNotch.

[0145] Therefore, the SynNotch system designed in this invention, which contains the transmembrane region of the Notch receptor protein derived from Xenopus tropicalis, has stronger activation efficiency than the SynNotch receptor system containing the transmembrane region of the original Notch receptor protein derived from mouse.

[0146] Example 3: Activation of Jurkat T cells with SynNotch-CAR gated chimeric antigen receptor by target cancer cells

[0147] Currently, T cells expressing chimeric antigen receptors (CARs) are effective in treating certain B-cell cancers. However, one of the major problems with CAR-T cell cancer immunotherapy is off-target effects, which can lead to the destruction of normal tissues by therapeutic CAR-T cells, resulting in serious side effects and even death. A potential approach to alleviate this problem is to enable more precise tumor targeting of therapeutic CAR-T cells. Expressing CARs that recognize an additional tumor antigen would provide a more precise T cell response. To achieve this, we hypothesize that the SynNotch synthetic receptor can be used to engineer therapeutic CAR-T cells that first bind to a tumor-specific cell surface antigen to detect tumors and then target the second tumor-specific antigen in the tumor for CAR expression, thereby providing dual-antigen control of CAR-T cell activity and tumor-localized responses.

[0148] Based on this, the inventors have conducted corresponding experiments with Jurkat cells (T cell line) to provide a synthetic antibody containing SynNotch-CAR gated chimeric antigen receptor (SynNotch-CAR-Jurkat T cells), specifically comprising the following steps: (1) SynNotch synthetic receptor 1 in Example 2 was subjected to slow viral packaging, and the slow viral packaging was capable of recognizing the CD19 CAR molecule. The nucleic acid encoding the CAR molecule contained a UAS-minimal-CMV sequence at the 5' end, which binds to an intracellular polypeptide produced after activation of the SynNotch synthetic receptor, inducing the expression of the CAR molecule (synthesized directly by Kin Weizhi Company and subjected to slow viral packaging; the CAR molecule had the amino acid sequence shown in SEQ ID NO: 10 and the nucleotide sequence shown in SEQ ID NO: 11; hereinafter, the CAR molecule is referred to as an inducible CAR molecule). Jurkat T cells were simultaneously infected with the two viruses, and the Jurkat T cells were selected to stably express the SynNotch synthetic receptor and the inducible CAR molecule. (2) Using conventional methods in the field, K562 cells expressing membrane-anchored GFP alone, membrane-anchored CD19 alone (the structure is shown in Figure 5, where CD19 has the amino acid sequence shown in SEQ ID NO:12 and the nucleotide sequence shown in SEQ ID NO:13), and co-expressing membrane-anchored GFP and CD19 were constructed as transducing cells. (3) The three types of K562 cells from step (2) above were co-incubated with the Jurkat cells constructed in step (1). After 24 hours, the expression of CD69 and the secretion of IL-2 and TNF-α of Jurkat T cells in each group were detected. The results are shown in Figure 6. The expression of CD69 and the secretion of IL-2 and TNF-α indicate the activation of Jurkat T cells. As shown in Figure 6, SynNotch-CAR-Jurkat T cells are activated upon exposure to K562 expressing two antigens (GFP and CD19). MALPVTALLLPLALLLHAARPEQKLISEEDLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:10); METDTLLLWVLLLWVPGSTGDYPYDVPDYAGAQPARPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVDEQKLISEEDLNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR(SEQ ID NO:12);

[0149] Example 4: Killing of target cancer cells by NK cells combined with SynNotch-CAR gated chimeric antigen receptor (abbreviated as SynNotch-CAR-NK cells)

[0150] The SynNotch synthetic receptor 1 of the present invention is common not only to T cells but also to other types of immune cells. The inventors confirmed that the SynNotch-CAR system described in Example 4 also functions in other immune cells by applying it to NK cells, specifically, the following steps: (1) In Example 2, slow virus packaging was performed on the SynNotch synthetic receptor (SynNotch synthetic receptor 1 or SynNotch synthetic receptor 2). The slow virus packaging was capable of recognizing the CD19 CAR molecule. The nucleic acid sequence encoding the CAR molecule contained a single UAS-minimal-CMV sequence at the 5' end. This sequence binds to an intracellular polypeptide generated after SynNotch synthetic receptor activation, inducing the expression of the CAR molecule (synthesized directly by Kin Weizhi Company and packaged for slow virus packaging; the CAR molecule has the amino acid sequence shown in SEQ ID NO: 10 and the nucleotide sequence shown in SEQ ID NO: 11). NK cells were simultaneously infected with the two viruses, and NK cells stably expressing the SynNotch synthetic receptor and the CAR molecule were selected. NK cells stably expressing the SynNotch synthetic receptor and the CAR molecule (i.e., CAR-NK) were used as a positive killing control, and NK-EV (NK cells infected with a virus produced by a blank plasmid) served as a negative killing control. (2) Huh7 cells expressing membrane-anchored GFP alone, membrane-anchored CD19 alone (see step (2) in Example 3 for the specific sequences), and co-expressing membrane-anchored GFP and CD19 were constructed as transducing cells. (3) Label the transducing cells described in (2) with CTV dye (purchased from Thermo Fisher). (4) The three types of Huh7 cells from step (3) were co-incubated with the NK cells constructed in step (1). After 48 hours, the cells were harvested and dead cells were labeled with PI dye. The survival status of the Huh7 cells in each group was analyzed and determined. See Figure 7. The survival rate of Huh7 cells represents the killing status of the NK cells against the recipient cells. As shown in Figure 7, when exposed to Huh7 cells co-expressing two antigens (GFP and CD19), SynNotch-CAR-NK cells were significantly activated and exhibited a higher killing effect against Huh7 cells. Compared with SynNotch-CAR (M-Syn-CAR) containing SynNotch synthetic receptor 2, the killing efficiency of SynNotch-CAR (X-Syn-CAR) containing SynNotch synthetic receptor 1 was significantly improved. Furthermore, the NK cell killing efficiency of SynNotch-CAR containing SynNotch synthetic receptor 1 was not significantly different from that of NK cells stably expressing a CAR molecule (CAR-NK) that can recognize CD19.

[0151] In the description herein, references such as "one embodiment," "some embodiments," "examples," "specific examples," or "some examples" mean that the specific features, structures, materials, or characteristics described in combination with the embodiment or example are included in at least one embodiment or example of the present invention. In the description herein, the terms "exemplary" and "specific examples" do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Furthermore, if not in conflict with each other, those skilled in the art may combine and combine features of different embodiments or examples described herein.

[0152] Although embodiments of the present invention have been presented and described, the above embodiments are illustrative and should not be construed as limiting the present invention, and those skilled in the art will appreciate that various changes, modifications, substitutions and variations can be made to the above embodiments within the scope of the present invention.

Claims

1. Use of an isolated polypeptide in the preparation of a SynNotch synthetic receptor, characterized in that the isolated polypeptide has at least 80% identity to the transmembrane domain of a Notch receptor protein derived from Xenopus tropicalis.

2. A chimeric polypeptide comprising: an extracellular domain having an activity of binding to a first molecule; a transmembrane domain whose N-terminus is connected to the C-terminus of the extracellular domain; and an intracellular domain whose N-terminus is connected to the C-terminus of the transmembrane domain; A chimeric polypeptide, wherein the transmembrane domain comprises a separate polypeptide, the separate polypeptide having at least 80% identity with the transmembrane domain of a Notch receptor protein derived from Xenopus tropicalis.

3. The use of claim 1 or the chimeric polypeptide of claim 2, characterized in that the isolated polypeptide has 100% identity with the transmembrane domain of the Notch receptor protein from Xenopus tropicalis.

4. The use according to claim 1 or the chimeric polypeptide according to claim 2, characterized in that the transmembrane domain of the Notch receptor protein derived from Xenopus tropicalis has the amino acid sequence as shown in SEQ ID NO:

1.

5. The chimeric polypeptide of claim 2, wherein the first molecule comprises 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.

6. The chimeric polypeptide of claim 5, wherein the tumor antigen comprises at least one of a tumor-associated antigen and a tumor-specific antigen, and is preferably a tumor-specific antigen.

7. The first molecule may be 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, CD 40, CD44, CD44v6, CD45, CD48, CD51, CD52, CD56, CD59, CD66, CD70, CD71, CD72, CD73, CD74, CD79A, C D79B, CD80, CD86, CD94, CD95, CD133, CD134, CD140, CD152, CD154, CD158, CD178, CD181, CD182, CD1 83, CD200, CD210, CD221, CD246, CD252, CD253, CD261, CD262, CD273, CD274, CD276, CD279, CD295, C D339, CD340, EGFR, HER2, FGFR2, AFP, CA125, MSLN, GPC3, CEA, CLDN18.2, EpCAM, PSCA, GD2, IL-13, I 3. The chimeric polypeptide of claim 2, comprising at least one of, but not limited to, L-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.

8. The chimeric polypeptide of claim 2, wherein the extracellular region comprises a first binding protein or a fragment thereof that binds to the first molecule.

9. The chimeric polypeptide of claim 8, wherein the first binding protein or fragment thereof comprises at least one of an antibody or functional fragment thereof, a receptor, a receptor ligand, and a cell adhesion molecule.

10. The chimeric polypeptide of claim 8, wherein the first binding protein or fragment thereof is a monoclonal or polyclonal antibody that binds to the first molecule.

11. The chimeric polypeptide of claim 10, wherein the monoclonal antibody comprises at least one of a Fab antibody, an F(ab')2 fragment, an Fv antibody, a single-chain antibody, a single-domain antibody, and a minimal recognition unit.

12. The chimeric polypeptide of claim 2, characterized in that the intracellular region comprises at least one of a transcriptional activator protein, a transcriptional inhibitory protein, a transcription factor, a site-specific nuclease, a recombinase, an activating immunoreceptor intracellular structural domain, and an inhibitory immunoreceptor intracellular structural domain.

13. The chimeric polypeptide of claim 12, wherein the intracellular region comprises at least one of GaL4-VP64, GaL4-VP16, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, HAP1-VP16, LexA-VP64, Cas9, and Cas13.

14. The chimeric polypeptide of claim 13, wherein the Gal4-VP64 has the amino acid sequence shown in SEQ ID NO:

2.

15. the transmembrane and intracellular domains have the amino acid sequences set forth in SEQ ID NO: 3; and / or The chimeric polypeptide of claim 2, wherein the chimeric polypeptide has the amino acid sequence shown in SEQ ID NO:

4.

16. A first nucleic acid molecule, wherein the first nucleic acid molecule encodes the chimeric polypeptide of any one of claims 2 to 15; A first nucleic acid molecule, characterized in that the nucleic acid molecule is DNA.

17. A first expression vector, characterized in that it has the first nucleic acid molecule of claim 16.

18. 18. The first expression vector according to claim 17, wherein the first expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage, preferably a plasmid expression vector.

19. 1. A recombinant cell comprising: A first nucleic acid molecule according to claim 16 or a first expression vector according to any one of claims 17 to 18, or A recombinant cell characterized by expressing the chimeric polypeptide according to any one of claims 2 to 15.

20. The recombinant cell is obtained by introducing the first expression vector according to any one of claims 17 to 18 into a host cell, 20. The recombinant cell of claim 19, wherein the host cell comprises at least one of an immune cell, a neuron, a progenitor or progenitor cell, an epithelial cell, an endothelial cell, and a stem cell.

21. 21. The recombinant cell of claim 20, wherein the host cell comprises at least one of a T cell, a B cell, a monocyte, a NK cell, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell, a cytotoxic T cell, a NKT cell, and a γδT cell.

22. The recombinant cell further comprises:

20. The recombinant cell of claim 19, further comprising a second nucleic acid molecule encoding a chimeric antigen receptor, NK cell receptor, or T cell receptor, or a second expression vector carrying said second nucleic acid molecule, or expressing said chimeric antigen receptor, NK cell receptor, or T cell receptor.

23. The recombinant cell of claim 22, wherein the intracellular region of the chimeric polypeptide comprises 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 nucleic acid sequence, which is used to bind to the transcriptional activator protein.

24. A pharmaceutical composition comprising the chimeric polypeptide of any one of claims 2 to 15, the first nucleic acid molecule of claim 16, the first expression vector of any one of claims 17 to 18, or the recombinant cell of any one of claims 19 to 23, Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

25. Use of the chimeric polypeptide according to any one of claims 2 to 15, the first nucleic acid molecule according to claim 16, the first expression vector according to any one of claims 17 to 18, the recombinant cell according to any one of claims 19 to 23 or the pharmaceutical composition according to claim 24 in the preparation of a medicament for preventing and / or treating a disease, comprising: Optionally, the disease comprises cancer or tumor, autoimmune disease, inflammation and diseases associated with cellular senescence.

26. 25. The chimeric polypeptide of any one of claims 2 to 15, the first nucleic acid molecule of claim 16, the first expression vector of any one of claims 17 to 18, the recombinant cell of any one of claims 19 to 23 or the pharmaceutical composition of claim 24 for preventing and / or treating a disease, optionally wherein the disease comprises cancer or tumor, autoimmune disease, inflammation and diseases associated with cellular senescence.

27. 1. A method for preventing and / or treating a disease, comprising: administering to a subject a pharmaceutically acceptable dose of the chimeric polypeptide of any one of claims 2 to 15, the first nucleic acid molecule of claim 16, the first expression vector of any one of claims 17 to 18, the recombinant cell of any one of claims 19 to 23, or the pharmaceutical composition of claim 24; Optionally, the method for preventing and / or treating a disease, characterized in that the disease comprises cancer or tumor, autoimmune disease, inflammation and diseases associated with cellular senescence.

28. 1. A method for activating immune cells, comprising: A method for activating an immune cell, comprising a step of first contacting the immune cell with a first molecule, wherein the immune cell expresses a chimeric polypeptide described in any one of claims 2 to 15.

29. the intracellular domain of the chimeric polypeptide comprises a transcriptional activator protein, and the immune cell-expressed chimeric antigen receptor, NK cell receptor, or T cell receptor, the chimeric antigen receptor, NK cell receptor, or T cell receptor comprises an antibody or a functional fragment thereof that binds to a predetermined antigen, the 5' end of the second nucleic acid molecule encoding the chimeric antigen receptor, NK cell receptor, or T cell receptor is connected to an inducible expression nucleic acid sequence, and the inducible expression nucleic acid sequence is used to bind to the transcriptional activator protein; The method comprises: after said first contact, said immune cell releases said transcriptional activator protein and expresses said chimeric antigen receptor, NK cell receptor or T cell receptor; and further comprising a step of binding a chimeric antigen receptor, an NK cell receptor, or a T cell receptor of the immune cell to the predetermined antigen, thereby activating the immune cell; 29. The method of claim 28, wherein optionally, the immune cells comprise 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.

30. 1. A method for tracing first cell-second cell contact, comprising: a step of performing a second contact between the first cell and the second cell, wherein the first cell expresses the chimeric polypeptide of any one of claims 2 to 15 and a reporter gene protein, the intracellular domain of the chimeric polypeptide is a transcriptional activator protein, the 5' end of a 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 transcriptional activator protein, and the second cell expresses the first molecule; A method for tracking contact between a first cell and a second cell, comprising a step of determining the contact status between the first cell and the second cell based on the detection result of the reporter gene protein in the first cell.

31. the first cell or the second cell comprises at least one of an immune cell, a neuron, a progenitor or precursor cell, an epithelial cell, an endothelial cell, and a stem cell; 31. The method of claim 30, wherein optionally, the first cell comprises at least one of a T cell, a B cell, a monocyte, a NK cell, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell, a cytotoxic T cell, a NKT cell, and a γδT cell.

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