SynNotch receptor and its use

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] This subfield belongs to the field of biotechnology. Specifically, the present invention relates to SynNotch receptors and their uses. 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 their uses, methods for activating immune cells, and methods for tracking the contact between a first cell and a second cell.

Background Art

[0002] With the development of synthetic biology, cells are modified to be able to recognize specific extracellular signals, and the recognized signals are transmitted to the modified cells, making artificially designed reactions more practical and applicable in the field of biomedicine. For example, in addition to the application of chimeric antigen receptor (CAR) technology in clinical treatment, in 2016, the Wendell A. Lim team disclosed a set of SynNotch receptor systems based on the Notch signaling pathway. This system can also recognize cell membrane surface ligands, and its signal output is not limited to simple immune cell activation signals, but can achieve various different signal output types, such as activation and suppression of the expression of specific genes. This receptor system can realize various combinations of different input signals and output signals, has orthogonality between multiple groups of systems within the same system (cells), and at the same time, can operate in many cells, has universality, and at the same time has many excellent characteristics such as simplicity and controllability. It is a powerful tool for modifying cells by applying synthetic biology.

[0003] In biology, the conventional Notch signaling pathway (system) includes the ligand corresponding to Notch, the Notch molecule, and downstream genes that regulate Notch. The Notch molecule is a transmembrane molecule and is divided into three parts: an extracellular structure, a transmembrane domain, and an intracellular structure domain. After the extracellular structure domain binds to the corresponding Notch ligand, the cell where the ligand is located undergoes engulfment activity, pulling the Notch molecule and exposing the S2 protease cleavage site on the extracellular structure domain of the Notch molecule. This site is then cleaved by members of the metalloprotease ADAMs family, and further, following the cleavage of the extracellular structure domain, the intracellular structure domain of the Notch molecule is cleaved by γ-secreting enzymes and released into the cell. Subsequently, it enters the nucleus and regulates the expression of downstream related genes as a transcription factor. The Wend ell A. Lim team modified the mouse Notch1 receptor by replacing its extracellular domain with a single-chain antibody or nanoantibody, and its intracellular domain with a transcriptionally activating or repressive structural domain, leaving only a transmembrane domain that can be recognized and cleaved by proteases, and also deploying downstream controlled gene elements. For different targets, the extracellular domain employs a single-chain antibody that specifically recognizes the antigen, and the intracellular domain controls the expression of a pre-defined target gene or factor. In the same cell, it is possible to design gene regulatory loops that are activated by different antigens and have good orthogonality. The SynNotch system includes many cell types, such as nerve cells, tumor cells, epidermal cells, and immune cells. CAR technology and the SynNotch system can be applied in combination to modify immune cells, enabling "AND gate" activation of immune cells, that is, immune cells can only be activated when the cell simultaneously expresses two specific surface antigens. By altering the gene elements controlled downstream of SynNotch, immune cells secrete therapeutic factors such as single-chain antibodies and cytokines after contact with specific antigens, and these factors exhibit antitumor effects both in vitro and intracellularly. This demonstrates that the SynNotch receptor system offers a significant technological advantage in cell remodeling. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] The present invention aims to solve, to at least to some extent, one of the technical problems present in the prior art. To this end, the present invention provides a SynNotch receptor that can enhance the ability of downstream genes to activate and reduce background leak activation levels.

[0005] This invention is completed based on the following statements of the inventors.

[0006] While the SynNotch system is highly functional, the commonly used early version of the SynNotch system (also known as the SynNotch synthesis receptor or SynNotch) has the following drawbacks: 1. After activation, the early version of SynNotch does not have a very strong ability to regulate downstream transcriptional expression; that is, the expression level required to activate downstream genes is not very high, limiting the use of SynNotch, especially in cases where downstream genes must be highly expressed to be effective. 2. The early version of SynNotch has a high background leak level, and for genes that require strict downstream control, this high level of leak expression can lead to the deactivation of the SynNotch molecular switch, causing the molecular expression pathway to bypass the SynNotch molecular switch directly.

[0007] However, through experimentation, the inventors unexpectedly discovered that the above problem stemmed from a deficiency in the transmembrane domain of the SynNotch molecule. The initial version of the SynNotch synthesis receptor selects the transmembrane domain of the mouse-derived Notch molecule as its transmembrane domain. Considering the differences in the S2 and S3 cleavage sequences carried by Notch transmembrane domains from different genera, and the conformational and sequence preferences present in S2 and S3 cleavage enzymes from different species, SynNotch synthesis receptors constructed with different transmembrane domains inevitably exhibit differences in their activation and leak efficiency in cells of different species. With the ultimate goal of modifying human-derived cells with the SynNotch synthesis receptor, the inventors unexpectedly selected the African clawed frog Notch transmembrane domain during the experimental process. This increased the transcriptional activation ability and efficiency of the SynNotch synthesis receptor to downstream genes, and reduced its background leak activation, allowing the SynNotch synthesis receptor to perform its role more effectively. [Means for solving the problem]

[0008] Therefore, in one aspect of the present invention, the present invention proposes the use of isolated polypeptides in the preparation of a SynNotch synthesis receptor, the isolated polypeptides having at least 80% identity with the transmembrane region of the Notch receptor protein derived from the tropical frog (Xenopus tropicalis). The inventors have found that a SynNotch synthesis receptor prepared by selectively preparing the transmembrane region of the tropical frog Notch receptor protein can enhance the transcriptional activation ability and efficiency of downstream genes and reduce background leak of the SynNotch synthesis receptor.

[0009] In another aspect of the present invention, the present invention proposes a chimeric polypeptide. According to an embodiment of the present invention, the chimeric polypeptide comprises an extracellular domain having activity to bind 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 separated polypeptide having at least 80% identity with the transmembrane domain of the Notch receptor protein derived from the tropical clawed frog. By selecting the transmembrane domain of the tropical clawed frog Notch receptor protein as the transmembrane domain of the chimeric polypeptide (i.e., SynNotch synthesis 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 leak of the chimeric polypeptide.

[0010] In another aspect of the present invention, the present invention proposes a first nucleic acid molecule. According to an example of the present invention, the first nucleic acid molecule encodes the chimeric polypeptide described in the claim. The first nucleic acid molecule according to an example of the present invention encodes the chimeric polypeptide.

[0011] In another aspect of the present invention, the present invention proposes a first expression vector. According to an embodiment of the present invention, the first expression vector has the first nucleic acid molecule described above. After introducing appropriate receptor cells into the first expression vector of the present invention, the expression of the chimeric polypeptide described above can be achieved.

[0012] In another aspect of the present invention, the present invention proposes recombinant cells. According to the embodiments of the present invention, the recombinant cells possess the aforementioned first nucleic acid molecule or the aforementioned first expression vector, or express the aforementioned chimeric polypeptide. The recombinant cells of the present invention can effectively express the aforementioned chimeric polypeptide.

[0013] In another aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the aforementioned chimeric polypeptide, the aforementioned first nucleic acid molecule, the aforementioned first expression vector, or the aforementioned recombinant cell. As can be seen from the above, a cell expressing the chimeric polypeptide can recognize the first molecule and, after binding to the first molecule, can achieve various types of signal output, such as activation and suppression of the expression of a specific gene, or, after contact with the first molecule using an immune cell expressing the chimeric polypeptide, can secrete a therapeutic factor (e.g., single-chain antibody, cytokine, etc.) and be used for antitumor purposes. Furthermore, the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in cells (e.g., immune cells), and can express the chimeric polypeptide on the aforementioned recombinant cell. For this reason, by employing a pharmaceutical composition comprising the aforementioned chimeric polypeptide, the first nucleic acid molecule, the first expression vector, or recombinant cell, the first molecule can be targeted and used for the prevention and / or treatment of related diseases, such as the treatment of cancer.

[0014] In another aspect of the present invention, the present invention proposes the use of the aforementioned chimeric polypeptide, the aforementioned first nucleic acid molecule, the aforementioned first expression vector, the aforementioned recombinant cell, or the aforementioned pharmaceutical composition in the preparation of drugs for the prevention and / or treatment of disease. As can be seen from the above, cells expressing the above chimeric polypeptide can recognize the first molecule and, after binding to the first molecule, can achieve various different signal output types, such as activation and suppression of the expression of specific genes, or, after contact with the first molecule using immune cells expressing the chimeric polypeptide, they can secrete therapeutic factors (e.g., single-chain antibodies, cytokines, etc.) and be used for antitumor 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 on the aforementioned recombinant cells. The pharmaceutical composition comprises the above-mentioned chimeric polypeptide, the first nucleic acid molecule, the first expression vector, or recombinant cells, and thereafter, a drug comprising the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, recombinant cells, or the pharmaceutical composition can be used to target the first molecule and for the prevention and / or treatment of related diseases such as cancer.

[0015] In another aspect of the present invention, the present invention proposes the aforementioned chimeric polypeptide, the aforementioned first nucleic acid molecule, the aforementioned first expression vector, the aforementioned recombinant cells, or the aforementioned pharmaceutical composition, which can be used for the prevention and / or treatment of diseases. As can be seen from the above, cells expressing the above chimeric polypeptide can recognize the first molecule and, after binding to the first molecule, can achieve various different signal output types, such as activation and suppression of the expression of specific genes, or, after contact with the first molecule using immune cells expressing the chimeric polypeptide, can secrete therapeutic factors (e.g., single-chain antibodies, cytokines, etc.) and be used for antitumor purposes. Furthermore, the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in cells (e.g., immune cells), and can express the chimeric polypeptide on the aforementioned recombinant cells. The pharmaceutical composition comprises the aforementioned chimeric polypeptide, the first nucleic acid molecule, the first expression vector, or recombinant cells, thereby allowing drugs comprising the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, recombinant cells, or pharmaceutical composition to target the first molecule and be used for the prevention and / or treatment of related diseases, such as the treatment of cancer.

[0016] In another aspect of the present invention, the present invention proposes a method for treating or preventing a disease. According to an embodiment of the present invention, the method comprises the step of administering to a subject a pharmaceutically acceptable amount of the aforementioned recombinant cells or the aforementioned pharmaceutical composition. 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 suppression of the expression of specific genes, or, after contact with the first molecule using immune cells expressing the chimeric polypeptide, can secrete therapeutic factors (e.g., single-chain antibodies, cytokines, etc.) that can be used for antitumor 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 on the aforementioned recombinant cells. The pharmaceutical composition comprises the aforementioned chimeric polypeptide, the first nucleic acid molecule, the first expression vector, or recombinant cells. Thus, a drug comprising the chimeric polypeptide, recombinant cells, or pharmaceutical composition can be used to target the first molecule for the prevention and / or treatment of related diseases such as cancer.

[0017] In another aspect of the present invention, the present invention proposes a method for activating immune cells. According to an embodiment of the present invention, the method comprises the step of making a first contact between the immune cells and a first molecule, wherein the immune cells express the aforementioned chimeric polypeptide. As a result, the immune cells expressing the chimeric polypeptide can recognize the first molecule and, after binding to the first molecule, can achieve activation of the immune cells.

[0018] In another aspect of the present invention, the present invention proposes a method for tracking contact between a first cell and a second cell. According to an embodiment of the present invention, the method comprises the step of making a second contact between the first cell and the second cell, wherein the first cell expresses the aforementioned chimeric polypeptide and reporter gene protein, the intracellular region of the chimeric polypeptide is a transcription-activating 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 transcription-activating protein, the second cell expresses the first molecule, and the contact status between the first cell and the second cell is determined based on the detection result of the reporter gene protein in the first cell. Thus, by employing the above method, it is possible to determine whether or not there is contact between the first cell and the second cell based on the detection result of the reporter gene protein.

[0019] Additional aspects and advantages of the present invention are partially shown in the following description, partially become apparent from the following description, or can be understood through the practice of the present invention. [Brief explanation of the drawing]

[0020] The above and / or additional aspects and advantages of the present invention will become apparent and easier to understand from the description of the embodiments combined with the following drawings. [Figure 1] This is a schematic diagram of the structure of the SynNotch synth receptor in Example 1 of the present invention. [Figure 2] This is a schematic diagram illustrating how SynNotch synthesis receptor cells recognize specific extracellular signals in Example 2 of the present invention. [Figure 3] This is a schematic diagram of the membrane-immobilized GFP structure in Example 2 of the present invention. [Figure 4] This shows the leak efficiency and effective activation efficiency of the mouse SynNotch synthesis receptor containing the Notch transmembrane structure domain and the Xenopus tropicalis SynNotch synthesis receptor containing the Notch transmembrane structure domain in Example 2 of the present invention. [Figure 5]It is a schematic diagram of the structure of membrane-fixed CD19 in Example 3 of the present invention. [Figure 6] It is the activation result of target cancer cells against SynNotch-CAR-Jurkat T cells in Example 3 of the present invention. [Figure 7] It is the killing efficiency of SynNotch-CAR-NK cells against target cancer cells in Example 4 of the present invention.

Mode for Carrying Out the Invention

[0021] Hereinafter, examples of the present invention will be described in detail. The examples described below are exemplary and are used only for interpreting the present invention, and are not intended to limit the present invention.

[0022] In addition, the terms "first" and "second" are used only for the purpose of explanation and cannot be understood as indicating or implying relative importance or suggesting the number of technical features shown. Therefore, the features limited by "first" and "second" can explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, for example, two, three, etc., unless otherwise specifically limited.

[0023] In this specification, the terms "comprising" or "including" are restrictive expressions, that is, they include the content shown by the present invention, but do not exclude the content of other aspects.

[0024] In this specification, the terms "optionally", "any" or "arbitrary" usually mean that the events or situations described below may occur, but do not necessarily occur, and the description includes both the cases where the events or situations occur and the cases where the events or situations do not occur.

[0025] In this specification, the terms “identity,” “homology,” or “similarity” refer to the determination of the proportion of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences in conventional methods when describing an amino acid sequence or nucleic acid sequence relative to a reference sequence, for example, 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). See Foundation, Washington, DC. Sequence comparison and sequence identity measurement 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, including but 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., as well as FASTA and TFASTA, which are available in the Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA, and in CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0026] In this specification, the term "at least 80% identity" means having at least 80% identity with each reference sequence, and may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0027] In this specification, the term “expression vector” typically refers to a nucleic acid molecule that can be inserted into a suitable host and self-replicate, and that moves the inserted nucleic acid molecule into and / or between host cells. The expression vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for duplication of DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The expression vector may further include vectors having a variety of the above functions. The expression vector may also be a polynucleotide that can be transcribed into a polypeptide and translated upon introduction into a suitable host cell. Typically, the expression vector can produce the desired expression product by culturing a suitable host cell containing the expression vector.

[0028] In this specification, the term “recombinant cell” typically refers to cells with unique traits and stable inheritance obtained by modifying, recombining, or otherwise altering 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. As used herein, the terms “transformation” or “transfect” refer to the introduction of nucleic acids (e.g., vectors) into a cell using various techniques known in the art. A suitable host cell can be transformed or transfected with the DNA sequence of the present invention and used for the expression and / or secretion of a target protein.

[0029] In this specification, the term “pharmaceutical composition” usually refers to a unit dose form and can be prepared by any method well known in the pharmaceutical field. All methods involve a step of conjugating an active ingredient with a vector comprising one or more accessory components. Typically, compositions are prepared by uniformly and sufficiently conjugating an active chimeric polypeptide or recombinant cells with a liquid vector, a shredded solid vector, or both.

[0030] In this specification, the term “pharmaceutically acceptable excipient” may include any solvent, solid excipient, diluent or other liquid excipient, suitable for a specific target dosage form. This is also considered by the present invention, except in cases where conventional excipients are incompatible with the chimeric polypeptide or recombinant cells of the present invention, for example, if they result in any undesirable biological effects or interact in a detrimental manner with any other component of the pharmaceutically acceptable composition.

[0031] In this specification, 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, provided that they can reach the desired tissue. Various methods of administration are possible, but the present invention is not limited to these exemplary methods of administration, such as peritoneal, intravenous, intramuscular, and subcutaneous injections. Preferably, the compositions of the present invention are administered by intravenous or subcutaneous injection.

[0032] In this specification, the term “treatment” means used to obtain a desired pharmacological and / or physiological effect. Such effect may be prophylactic in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it partially or completely cures a disease and / or the adverse effects caused by a disease. As used herein, “treatment” covers diseases of mammals, particularly humans, and includes (a) preventing disease or the onset of disease in individuals that are susceptible to disease but have not yet been diagnosed with the disease, (b) suppressing disease, such as by hindering the progression of the disease, or (c) alleviating disease, for example, reducing symptoms associated with the disease. As used herein, “treatment” includes, but is not limited to, any administration of a chimeric polypeptide, recombinant cell, pharmaceutical composition or drug to an individual to treat, cure, alleviate, improve, reduce or suppress a disease in that individual, and includes, but is not limited to, administering a drug containing a chimeric polypeptide, recombinant cell or pharmaceutical composition described herein to an individual in need.

[0033] This invention proposes the use of SynNotch synthetic receptors in preparation, chimeric polypeptides, a first nucleic acid molecule, a first expression vector, recombinant cells, pharmaceutical compositions and their use, a method for activating immune cells, and a method for tracking contact between a first cell and a second cell, each of which will be described in detail below.

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

[0035] In one aspect of the present invention, the present invention proposes the use of an isolated polypeptide in the preparation of a SynNotch synthesis receptor, the isolated polypeptide having at least 80% identity with the transmembrane region of the Notch receptor protein derived from the tropical frog (Xenopus tropicalis). The inventors have found that a SynNotch synthesis receptor prepared by selectively using the transmembrane region of the tropical frog Notch receptor protein can enhance the transcriptional activation ability and efficiency of downstream genes and reduce background leak of the SynNotch synthesis receptor.

[0036] According to an embodiment of the present invention, the isolated polypeptide has 100% identity with the transmembrane region of the Notch receptor protein derived from the tropical frog (Xenopus tropicalis).

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

[0038] Chimeric polypeptide

[0039] In another aspect of the present invention, the present invention proposes a chimeric polypeptide. According to an embodiment of the present invention, the chimeric polypeptide comprises an extracellular region having activity to bind to a first molecule, a transmembrane region whose N-terminus is connected to the C-terminus of the extracellular region, and an intracellular region whose N-terminus is connected to the C-terminus of the transmembrane region, wherein the transmembrane region comprises a separated polypeptide having at least 80% identity with the transmembrane region of the Notch receptor protein derived from the tropical clawed frog.

[0040] The inventors discovered that by selecting the transmembrane region of the Notch receptor protein of the tropical clawed frog as the transmembrane region of a chimeric polypeptide (i.e., SynNotch synthesis receptor), the ability and efficiency of transcriptional activation of downstream genes by the chimeric polypeptide can be enhanced, and background leak of the chimeric polypeptide can be reduced. Furthermore, cells expressing the chimeric polypeptide can recognize the first molecule and, after binding to the first molecule, can achieve various types of signal output, such as activation and suppression of the expression of specific genes, or can be used to prepare immune cells expressing the chimeric polypeptide, which, after contact with the first molecule, can secrete therapeutic factors (e.g., single-chain antibodies, cytokines, etc.) and can be used for antitumor purposes.

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

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

[0043] According to embodiments of the present invention, the transmembrane region further comprises epidermal growth factor-like repeat sequences (EGF repeats) and / or RAM sequences. This further reduces background leak activation of the SynNotch synthesis 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 an amino acid sequence as shown in SEQ ID NO:16. EHGQLWFP (SEQ ID NO: 16).

[0046] According to embodiments of the present invention, the C-terminus of the epidermal growth factor-like repeat sequence is connected to the N-terminus of the isolated polypeptide, and / or the C-terminus of the isolated polypeptide is connected to the N-terminus of the RAM sequence.

[0047] According to embodiments 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 for a cell receptor.

[0048] In this specification, the term "tumor antigen" generally refers to antigenic substances that newly appear or are overexpressed during the development and growth of a tumor. Tumor antigens are classified into tumor-specific antigens and tumor-associated antigens according to tumor antigen-specific classification. Tumor-specific antigens (TSAs) are novel antigens that are unique to tumor cells or present only in certain tumor cells and not in normal cells, while tumor-associated antigens (TAAs) are antigens unique to non-tumor cells but also present in normal cells and other tissues, and their content is clearly higher during cell carcinogenesis. These include, but are not limited to, PD-L1, PD-1, TGF-β, CEA, GD2, and GD3.

[0049] In this specification, the terms “cell receptor” or “receptor” should be understood in a broad sense, referring to molecules located on the cell membrane that can recognize and bind to various extracellular signaling molecules (ligands), including, but not limited to, growth factor receptors (e.g., VEGF receptor), NKG2D polypeptide (receptor for MICA, MICB, and ULB6), cytokine receptors (e.g., IL-13 receptor, IL-2 receptor, etc.), epidermal growth factor (EGF) receptor, Her2, CD27, innate cytotoxic receptors (NCR) (e.g., NKP30 (NCR3 / CD337) polypeptide (receptor for HLA-B related 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), immunomodulatory receptors (positive coreceptors (e.g., CD28) and negative (immunosuppressive) coreceptors (e.g., PD1)), and receptors for immunomodulatory molecules (e.g., TGFβ).

[0050] In this specification, the term “ligand of a cell receptor” should be understood in a broad sense, referring to a chemical substance that can bind to and interact with cell membrane receptors and produce specific biological effects, such as polypeptides, nucleic acids, glycoproteins, small molecules, carbohydrates, lipids, glycolipids, lipoproteins, lipopolysaccharides, etc., and including, but not limited to, cytokines (e.g., IL-13), growth factors (e.g., heregulin, vascular endothelial growth factor (VEGF)), peptide hormones, integrin-binding peptides (e.g., peptides containing the sequence Arg-Gly-Asp), and N-polysaccharides.

[0051] For example, the ligand is VEGF and the receptor is the VEGF receptor, or the ligand is heregulin and the receptor is Her2.

[0052] In this specification, the term "cytokine" should be understood in a broad sense, referring to proteins or small polypeptide molecules that transmit information between cells and have immunomodulatory and efficacy functions, such as IL-10. The term "cytokine receptor" should also be understood in a broad sense, referring 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 present invention, the tumor antigen is a tumor-specific antigen.

[0054] According to embodiments 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 It includes at least one of the following: 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 embodiments of the present invention, the extracellular region comprises a first binding protein or a fragment thereof that binds to the first molecule, According to embodiments of the present invention, the first binding protein or fragment thereof comprises at least one of an antibody or a functional fragment thereof, a receptor, a ligand for a receptor, and a cell adhesion molecule.

[0056] In this specification, “cell adhesion molecule” or “CAM” may refer to polypeptides of components or binding cell surface molecules that bind to the extracellular matrix (ECM). For example, the cell adhesion molecule may be the extracellular domain of CAM, CAM may be a calcium-independent adhesion molecule, for example, CAM is an immunoglobulin superfamily CAM, CAM may further be a calcium-dependent adhesion molecule, for example, CAM may be an integrin, cadherin or selectin, the cell adhesion molecule may be an integrin, for example, a cadherin such as E-cadherin, P-cadherin, N-cadherin, R-cadherin or M-cadherin, and the cell adhesion molecule may be a selectin such as E-selectin, L-selectin or P-selectin.

[0057] In this specification, the term "antibody" is used in its broadest sense and may 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. Typically, an antibody molecule consists of a light chain with a low molecular weight and a heavy chain with a high molecular weight, and the heavy chain (H chain) and light chain (L chain) are connected by disulfide bonds. The amino acid sequence at the amino end (N end) of the peptide chain changes significantly and is called the variable region (V region), while the carboxyl group end (C end) is relatively stable and changes less 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] In this specification, the terms “full-length antibody,” “full-length monoclonal antibody,” or “full-length monoclonal antibody” all refer to antibodies consisting of at least two identical light chains and at least two identical heavy chains linked by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE).

[0059] In this specification, 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 possessing performance activity that allows it to specifically bind to an antigen. For example, the fragment contains part or all of an antibody CDR. Such fragments are biologically active, can bind to an antigen, and can compete with other antigen-binding molecules (including complete antibodies) to bind 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 antibody or polyclonal antibody that binds to the first molecule.

[0061] In this specification, the term "polyclonal antibody" is synonymous with "multispecific antibody," and both refer to antibodies capable of recognizing multiple antigen epitopes. For example, an antibody capable of recognizing two antigen epitopes (bispecific antibody, abbreviated as bi-antibody), an antibody capable of recognizing three antigen epitopes, or an antibody capable of recognizing four antigen epitopes. It should be understood in a broad sense, and the specific structure is not limited; it is sufficient that 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 embodiments of the present invention, 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 the smallest recognition unit.

[0063] In this specification, the terms “single-domain antibody,” “nano-antibody,” and “VHH antibody” are interchangeable and refer to an antigen-binding immunoglobulin (variable) domain (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 is initially described as a “heavy-chain antibody” (i.e., “antibody lacking a light chain”), and which contains a heavy-chain variable region (VH) and conventional CH2 and CH3 regions, and specifically binds to an antigen protein (e.g., D-dimer) via the heavy-chain variable region.

[0064] In this specification, the terms “Fab antibody” or “Fab fragment” usually refer to an antibody or fragment containing only Fab molecules, consisting of a heavy chain of VH and CH1 and a complete light chain, connected by a single disulfide bond between the light and heavy chains.

[0065] In this specification, the terms “F(ab')2 antibody” or “F(ab')2 fragment” include two antigen-binding F(ab') moieties linked by a disulfide bond.

[0066] In this specification, the terms “Fv antibody” or “Fv fragment” typically refer to an antibody or fragment in which only the light chain variable region (VL) and heavy chain variable region (VH) are non-covalently linked, and which is the smallest functional fragment in which the antibody molecule leaves a complete antigen-binding site.

[0067] In this specification, 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 linked by a short-chain peptide.

[0068] In this specification, the terms "minimum recognition unit" and "MRU" both refer to an antibody or fragment consisting of only one CDR, whose molecular weight is sufficiently small to account for only about 1% of the complete antibody.

[0069] According to embodiments of the present invention, the intracellular region includes at least one of a transcription-activating protein, a transcription-inhibiting protein, a transcription factor, a site-specific nuclease, a recombinant enzyme, an activating immune receptor intracellular structural domain, and an inhibitory immune receptor intracellular structural domain.

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

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

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

[0073] According to an embodiment of the present 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 of the present invention, the present invention proposes a first nucleic acid molecule. According to an embodiment of the present invention, the first nucleic acid molecule encodes a chimeric polypeptide described in the claim. The first nucleic acid molecule according to an embodiment of the present invention can obtain the above chimeric polypeptide by encoding.

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

[0077] Those skilled in the art should understand that the first nucleic acid molecule referred to herein actually includes one or two complementary double helixes. For convenience, although only one strand is often shown herein, the other complementary strand is also disclosed. Furthermore, the molecular sequences in this invention include either DNA or RNA forms, and the disclosure of one implies the disclosure of the other.

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

[0079] In this specification, “manipulable connection” means that an exogenous gene is connected to a vector so that regulatory elements within the vector, such as transcriptional and translational regulatory sequences, can exert their function in regulating the transcription and translation of the desired exogenous gene. Commonly used vectors include plasmids and bacteriophages. In some specific embodiments of the present invention, after introducing appropriate receptor cells (also called receptor cells or host cells) into the vector, the expression of the aforementioned chimeric polypeptide can be effectively achieved via a regulatory system.

[0080] According to embodiments 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 embodiments of the present invention, the first expression vector is a plasmid expression vector.

[0082] In another aspect of the present invention, the present invention proposes recombinant cells. According to embodiments of the present invention, the recombinant cells possess the aforementioned first nucleic acid molecule or the aforementioned first expression vector, or express the aforementioned chimeric polypeptide. The aforementioned chimeric polypeptide can be effectively expressed within the recombinant cells under appropriate conditions using these recombinant cells.

[0083] Furthermore, the "appropriate conditions" described in this invention refer to conditions suitable for the expression of the aforementioned chimeric polypeptide. Those skilled in the art will readily understand that the conditions suitable for the expression of the aforementioned chimeric polypeptide include, but are not limited to, an appropriate transformation or transfect method, appropriate transformation or transfect conditions, healthy cell conditions, appropriate cell density, appropriate cell culture environment, and appropriate cell culture time. The "appropriate conditions" are not particularly limited, and those skilled in the art can optimize the conditions for optimal chimeric polypeptide expression according to the specific laboratory environment.

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

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

[0086] According to embodiments of the present invention, the host cell comprises at least one of the following: T cell, B cell, mononuclear cell, NK cell, dendritic cell, macrophage, regulatory T cell, helper T cell, cytotoxic T cell, NKT cell, and γδT cell.

[0087] According to embodiments of the present invention, the recombinant cells contain a second nucleic acid molecule encoding a chimeric antigen receptor, an NK cell receptor, or a T cell receptor, or a second expression vector having the second nucleic acid molecule, or discover the chimeric antigen receptor, T cell receptor, or NK cell receptor. The inventors' experiments have shown that the combination of the chimeric polypeptide of the present invention with a chimeric antigen receptor, an NK cell receptor, or a T cell receptor jointly modifies immune cells (e.g., Jurkat cells and NK cells), and that immune cells with the chimeric polypeptide + CAR / TCR combination can recognize the corresponding ligand, efficiently activate immune cells (e.g., Jurkat T cells and NK cells), and exert the function of killing targets (e.g., tumor cells).

[0088] In this specification, 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. “Chimeric antigen receptors (CARs)” are also called “chimeric receptors,” “T-isomers,” or “chimeric immune receptors (CIRs).” The “extracellular structural domain capable of binding to an antigen” refers to any oligopeptide or polypeptide capable of binding to a given antigen. The “intracellular structural domain” refers to any known oligopeptide or polypeptide that functions as a structural domain that transmits signals to activate or inhibit intracellular biological processes.

[0089] In this specification, the term “T cell receptor (TCR)” refers to a molecule found on the surface of T cells responsible for recognizing 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, the antigen is 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, and the binding of the TCR to the antigen peptide-MHC complex results in the activation of T lymphocytes, which express the TCR through a series of biochemical reactions mediated by relevant enzymes, co-receptors, and specialized accessory molecules on the T lymphocytes. The MHC molecules may be Class I or Class II MHC molecules. 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 of the gene complex that codes for the human major histocompatibility complex (MHC), and includes HLA type I antigens (A, B, and C) and HLA type II antigens (DP, DQ, and DR). HLA alleles A, B, and C primarily present peptides derived from intracellular proteins, such as proteins expressed within cells.

[0090] In this specification, the term "NK cell receptor" refers to molecules found on the surface of NK cells, which are divided into inhibitory receptors and activating receptors based on their mediating function. Inhibitory receptors recognize specific molecules expressed on the surface of normal cells and then transmit anti-killing signals to suppress their own killing function. There are many types of inhibitory receptors, including but not limited to CD161, CLRG1, PD1, TIM3, LAG3, CD96, and TIGIT. Activating receptors recognize corresponding ligands on the surface of target cells and then transmit activation signals into the cell to exert a killing effect. Activating receptors include but are not limited to NKp30, NKp44, NKp46, and CD16.

[0091] According to embodiments of the present invention, the intracellular region comprises a transcription-activating protein, the 5' end of a 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 transcription-activating protein.

[0092] For example, the intracellular region is GaL4-VP64, and the induced expression nucleic acid sequence is the UAS-minimal-CMV sequence.

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

[0094] In one preferred embodiment of the present invention, the chimeric antigen receptor, NK cell receptor, or T cell receptor has the activity to bind to a second molecule. For example, the chimeric antigen receptor binds to the second molecule mainly by its extracellular domain.

[0095] According to embodiments 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 for a cell receptor.

[0096] In one preferred embodiment of the present invention, the second 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, C It includes 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 of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the aforementioned chimeric polypeptide, the aforementioned first nucleic acid molecule, the aforementioned first expression vector, or the aforementioned recombinant cell. As can be seen from the above, a cell expressing the chimeric polypeptide can recognize the first molecule and, after binding to the first molecule, can achieve various types of signal output, such as activation and suppression of the expression of a specific gene, or, after contact with the first molecule using an immune cell expressing the chimeric polypeptide, can secrete a therapeutic factor (e.g., single-chain antibody, cytokine, etc.) and be used for antitumor purposes. Furthermore, the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in cells (e.g., immune cells), and can express the chimeric polypeptide on the aforementioned recombinant cell. For this reason, by employing a pharmaceutical composition comprising the aforementioned chimeric polypeptide, the first nucleic acid molecule, the first expression vector, or recombinant cell, the first molecule can be targeted and used for the prevention and / or treatment of related diseases, such as the treatment of cancer.

[0098] According to embodiments of the present invention, the invention further includes pharmaceutically acceptable adjuvants.

[0099] use

[0100] In another aspect of the present invention, the present invention proposes the use of the aforementioned chimeric polypeptide, the aforementioned first nucleic acid molecule, the aforementioned first expression vector, the aforementioned recombinant cell, or the aforementioned pharmaceutical composition in the preparation of drugs for the prevention and / or treatment of disease. As can be seen from the above, cells expressing the above chimeric polypeptide can recognize the first molecule and, after binding to the first molecule, can achieve various different signal output types, such as activation and suppression of the expression of specific genes, or, after contact with the first molecule using immune cells expressing the chimeric polypeptide, they can secrete therapeutic factors (e.g., single-chain antibodies, cytokines, etc.) and be used for antitumor 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 on the aforementioned recombinant cells. The pharmaceutical composition comprises the above-mentioned chimeric polypeptide, the first nucleic acid molecule, the first expression vector, or recombinant cells, and thereafter, a drug comprising the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, recombinant cells, or the pharmaceutical composition can be used to target the first molecule and for the prevention and / or treatment of related diseases such as cancer.

[0101] In another aspect of the present invention, the present invention proposes the aforementioned chimeric polypeptide, the aforementioned first nucleic acid molecule, the aforementioned first expression vector, the aforementioned recombinant cells, or the aforementioned pharmaceutical composition for preventing and / or treating diseases. As can be seen from the above, cells expressing the above chimeric polypeptide can recognize the first molecule and, after binding to the first molecule, can achieve various different signal output types, such as activation and suppression of the expression of specific genes, or, after contact with the first molecule using immune cells expressing the chimeric polypeptide, can secrete therapeutic factors (e.g., single-chain antibodies, cytokines, etc.) and be used for antitumor purposes. Furthermore, the first nucleic acid molecule and the first expression vector can express the chimeric polypeptide in cells (e.g., immune cells), and can express the chimeric polypeptide on the aforementioned recombinant cells. The pharmaceutical composition comprises the aforementioned chimeric polypeptide, the first nucleic acid molecule, the first expression vector, or recombinant cells, thereby allowing drugs comprising the chimeric polypeptide, the first nucleic acid molecule, the first expression vector, recombinant cells, or the pharmaceutical composition to target the first molecule and be used for the prevention and / or treatment of related diseases, such as the treatment of cancer.

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

[0103] According to embodiments of the present invention, the disease includes cancer or tumors, and immune-related diseases.

[0104] According to embodiments of the present invention, the disease includes cancer or tumors, autoimmune diseases, inflammation, and related diseases due to cellular senescence.

[0105] In this specification, the terms “cancer” or “tumor” may refer to uncontrolled cell growth. Examples include non-small cell lung cancer, papillary thyroid cancer, pleomorphic glial cell aneurysm, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, cholangiocarcinoma or sarcoma, acute osteomyelitis, large cell neuroendocrine carcinoma, neuroblastoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, squamous cell carcinoma of the head and neck, cervical cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma or gastric cancer.

[0106] method

[0107] In another aspect of the present invention, the present invention proposes a method for activating immune cells. According to an embodiment of the present invention, the method comprises the step of making a first contact between the immune cells and a first molecule, wherein the immune cells express the aforementioned chimeric polypeptide. As a result, the immune cells expressing the chimeric polypeptide can recognize the first molecule and, after binding to the first molecule, can achieve activation of the immune cells, which is particularly applicable to scientific research, and activated immune cells can be obtained by in vitro cell culture of the immune cells.

[0108] According to embodiments of the present invention, the intracellular region of the chimeric polypeptide comprises a transcription-activating protein, the immune cell expresses a 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 a second nucleic acid molecule encoding the chimeric antigen receptor, NK cell receptor, or T cell receptor is ligated to an inducibly expressed nucleic acid sequence, the inducibly expressed nucleic acid sequence is used to bind to the transcription-activating protein, and the method further comprises the steps of: after the first contact, the immune cell releases the transcription-activating protein and expresses the chimeric antigen receptor, NK cell receptor, or T cell receptor; and the immune cell activates the immune cell by binding the predetermined antigen to the chimeric antigen receptor, NK cell receptor, or T cell receptor of the immune cell.

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

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

[0111] In one preferred 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, C It includes 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 embodiments of the present invention, the immune cells include at least one of T cells, B cells, mononuclear cells, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells, and γδT cells.

[0113] In another aspect of the present invention, the present invention proposes a method for tracking contact between a first cell and a second cell. According to an embodiment of the present invention, the method comprises the step of making a second contact between the first cell and the second cell, wherein the first cell expresses the aforementioned chimeric polypeptide and reporter gene protein, the intracellular region of the chimeric polypeptide is a transcription-activating 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 transcription-activating protein, the second cell expresses the first molecule, and the contact status between the first cell and the second cell is determined based on the detection result of the reporter gene protein in the first cell. Thus, by employing the above method, it is possible to determine whether or not there is contact between the first cell and the second cell based on the detection result of the reporter gene protein, which is particularly applicable to scientific research, and the method can be used to determine whether or not there is contact between two types of cells in vitro.

[0114] According to an embodiment of the present invention, the inducible expression nucleic acid sequence has the nucleic acid sequence 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 soluble.

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

[0117] In one preferred 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, C It includes 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 embodiments of the present invention, the first or second cell comprises at least one of immune cells, neurons, primordial or progenitor cells, epithelial cells, endothelial cells, and stem cells.

[0119] According to embodiments of the present invention, the first cell comprises at least one of T cells, B cells, mononuclear cells, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells, and γδT cells.

[0120] In another aspect of the present invention, the present invention proposes a method for controlling cell activity. According to an embodiment of the present invention, the method comprises the step of making a third contact between the cell and a first molecule, wherein the cell expresses the aforementioned chimeric polypeptide.

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

[0122] According to embodiments of the present invention, the cells include at least one of T cells, B cells, mononuclear cells, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells, and γδT cells.

[0123] In another aspect of the present invention, the present invention proposes a method for promoting intracellular gene or protein expression. According to an embodiment of the present invention, the method comprises the step of making a fourth contact between the cell and a first molecule, wherein the cell expresses the aforementioned chimeric polypeptide, the cell has the gene, or expresses the protein, 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 embodiments of the present invention, the intracellular domain of the chimeric polypeptide is a transcription-activating protein, and the chimeric antigen receptor, second chimeric polypeptide, translational regulator, cytokine, hormone, chemokine, or antibody comprises a second binding protein or fragment thereof of the transcription-activating protein.

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

[0126] According to embodiments of the present invention, the cells include at least one of T cells, B cells, mononuclear cells, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells, and γδT cells.

[0127] In another aspect of the present invention, the present invention proposes a method for treating or preventing a disease. According to an embodiment of the present invention, the method comprises the step of administering to a subject a pharmaceutically acceptable amount of the aforementioned recombinant cells or the aforementioned pharmaceutical composition. 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 suppression of the expression of specific genes, or, after contact with the first molecule using immune cells expressing the chimeric polypeptide, can secrete therapeutic factors (e.g., single-chain antibodies, cytokines, etc.) that can be used for antitumor 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 on the aforementioned recombinant cells. The pharmaceutical composition comprises the aforementioned chimeric polypeptide, the first nucleic acid molecule, the first expression vector, or recombinant cells. Thus, a drug comprising the chimeric polypeptide, recombinant cells, or pharmaceutical composition can be used to target the first molecule for the prevention and / or treatment of related diseases such as cancer.

[0128] The effective dose of the recombinant protein or pharmaceutical composition described in the present invention can vary depending on the method of administration and the severity of the disease being treated. A preferred effective dose can be determined by those skilled in the art based on various factors (e.g., clinical trials). These factors include, but are not limited to, pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, and half-life; the severity of the disease the patient is trying to treat; the patient's body weight; the patient's immune status; and the route of administration. For example, depending on the urgent needs of the treatment situation, the drug may be administered in several divided doses per day or the dose may be proportionally reduced.

[0129] The recombinant proteins or pharmaceutical compositions of the present invention can be incorporated into drugs suitable for extra-gastrointestinal administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms, such as liquid, semi-solid, and solid dosage forms, and include, but are not limited to, liquid solutions (e.g., injectable solutions and infusion solutions) or lyophilized powders. Typical drugs are in the form of injectable or infusion solutions. The recombinant proteins or pharmaceutical compositions described above can be administered by intravenous infusion or injection, or by intramuscular or subcutaneous injection.

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

[0131] According to embodiments of the present invention, the disease includes cancer or tumors, immune-related diseases. According to embodiments of the present invention, the disease includes cancer or tumors, autoimmune diseases, inflammation, and related diseases due to cellular senescence.

[0132] According to embodiments of the present invention, the tumor or cancer includes, but is not limited to, non-small cell lung cancer, papillary thyroid cancer, pleomorphic glial cell aneurysm, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, cholangiocarcinoma or sarcoma, acute osteomyelitis, large cell neuroendocrine carcinoma, neuroblastoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, squamous cell carcinoma of the head and neck, cervical cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma or gastric cancer.

[0133] The means of this disclosure will be interpreted in combination with the following examples. Those skilled in the art will understand that the following examples are used solely to illustrate this disclosure and should not be considered to limit the scope of this disclosure. Where no specific technique or conditions are shown in the examples, they should be carried out in accordance with the techniques or conditions described in the art literature or in accordance with the product specifications. Where the manufacturer of the reagents or equipment used is not indicated, they are conventional products available on the market.

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

[0135] 1. The structure of the SynNotch synthesis receptor (abbreviated as SynNotch receptor) includes an extracellular domain (also called the extracellular region), a Notch transmembrane domain (also called the transmembrane region of the Notch receptor protein, abbreviated as the transmembrane region), and an intracellular domain (also called the intracellular region). The extracellular domain may be substituted with a single-chain antibody or nanoantibody, and the intracellular domain may be substituted with a structural domain that activates or represses transcription. In this invention, a modified SynNotch receptor was obtained by using an anti-GFP single-chain antibody as the extracellular domain portion, GAL4-VP64 as the intracellular domain portion, and a mouse or Xenopus tropicalis or Notch transmembrane domain that can be recognized and cleaved by a protease as the Notch transmembrane domain. The structure of the SynNotch receptor modified according to the design of this invention is shown, for example, in Figure 1. Figure 1 shows the structure of the SynNotch receptor including the Notch transmembrane domain (Notch core) of a mouse or Xenopus tropicalis.

[0136] 2. Construct the expression plasmid vector for the above SynNotch synthesis receptor. The specific steps are as follows, for example: (1) The Xenopus tropicalis SynNotch synthesis receptor containing the Notch transmembrane domain (abbreviated as SynNotch synthesis receptor 1) has the amino acid sequence shown in SEQ ID NO:4 and the nucleotide sequence shown in SEQ ID NO:5, and the mouse SynNotch synthesis receptor containing the Notch transmembrane domain (abbreviated as SynNotch synthesis receptor 2) has the amino acid sequence shown in SEQ ID NO:6 and the nucleotide sequence shown in SEQ ID NO:7. Next, Kim Yu-Ji Company synthesizes the nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:7, respectively, into the Puc57 vector. (2) The pCDH-EV vector is double digested using EocR1 and BamH1 endonucleases, followed by electrophoresis on a 1% agarose gel for 20 minutes, and then the double digested vector is recovered by rubber cutting. (3) Using primer pairs, nucleotide sequences as shown in SEQ ID NO:5 and SEQ ID NO:7 are obtained from the two pUC57 vectors of step (1) by PCR. Then, the nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:7 are homologously recombined with the nucleotide fragments of the SynNotch synthesis receptor and the double digestion vector obtained in step (2), respectively, 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 validated, and the sequencing results confirmed that the construction was successful. MALPVTALLLPLALLLHAARPEQKLISEEDLMADVQLVESGGGLVQAGGSLRLSCAASGRTISMAAMSWFRQAPGKEREFVAGISRSAGSAVHADSVKGRFTISRDNTKNTLYLQMNSLKAEDTAVYYCAVRTSGFFGSIPRTGTAFDYWGQGTQVTVSILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRRMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSAAAGGSGGSGGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGS(SEQ ID NO:6);

[0137] Example 2: Testing and validation of the SynNotch receptor at the cellular level.

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

[0139] This invention provides cellular-level verification of whether the expression plasmid vector (pCDH-antiGFP-XENTR-SynNotch-Gal4VP64) prepared in Example 1 enhances the ability of downstream gene transcriptional activation, using HT1080 cells as sender cells and similarly, HT1080 cells from a different lot as receiver cells. The specific verification steps are as follows. (1) The HT1080 cell line is infected with the slow virus and is capable of stably expressing membrane-fixed GFP, and is a transmission cell (the structure of membrane-fixed GFP is shown in Figure 3, for example, and GFP has an amino acid sequence as shown in SEQ ID NO:8 and a nucleotide sequence as shown in SEQ ID NO:9).

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

[0141] Using HT1080 cells from a different lot as receptor cells, we first infected them with the pHR_Gal4UAS_tBFP_PGK_mCherry slow virus (purchased from Addgene, #79130; this slow virus can induce BFP fluorescent protein expression by GAL4VP64 after infecting host cells). We then selected cells that could stably express high levels of pHR_Gal4UAS_tBFP_PGK_mCherry, and then infected them with the pCDH-antiGFP-XENTR-SynNotch-Gal4VP64 slow virus. pHR_Gal4UAS_tBFP_PGK_mCherry and pCDH-antiGFP-XENTR-SynNotch-Gal4VP64 slow viruses are obtained by co-transfecting the expression plasmid (pCDH-antiGFP-XENTR-SynNotch-Gal4VP64 or pHR_Gal4UAS_tBFP_PGK_mCherry), pMD2.G, and psPAX2 into a 15cm culture dish containing HEK293-T cells at a density of 90%, taking the cell supernatant after 3 days, and concentrating the virus by filtering the virus solution through a 0.45 μM filter. (2) Collect the above stably transfected receptor cells HT1080 cells, and set the cell density to 3 × 10 5 Inoculate into a 24-well plate at a rate of cells / mL, and the cell density will be 3 × 10⁶. 5 HT1080 cells expressing GFP at a rate of 1 / mL were added 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 shows the SynNotch synthesis receptor (abbreviated as SynNotch synthesis receptor 2) containing the mouse Notch transmembrane domain, i.e., the original system, while XENTR-Syn shows the Xenopus tropicalis SynNotch synthesis receptor (abbreviated as SynNotch synthesis receptor 1) containing the Notch transmembrane domain. As can be seen from Figure 4A, SynNotch synthesis receptor 1 significantly improves the efficiency of activation compared to SynNotch synthesis receptor 2.

[0143] Figure 4B shows the average 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 synthesis receptor 1 (i.e., XENTR group) is improved by 95.1% compared to SynNotch synthesis receptor 2 (i.e., MOUSE group), and the leak level of SynNotch synthesis receptor 1 (i.e., XENTR group) is reduced by 56.4%.

[0144] There are three forms of intercellular contact, including contact between wall-attached cells, contact between wall-attached cells and suspension cells, and contact between suspension cells. Since the three forms of contact also reflect to some extent the three true contact environments of cells in the body, it is important to confirm that all three cell contact methods can effectively activate the SynNotch system. On the other hand, HT1080 is a wall-attached cell, and the results in Figures 4A and 4B above can only prove that contact between wall-attached cells can activate the SynNotch system, but cannot be determined for the other two types of contact systems. For this reason, suspension receptor cells Jurkat and suspension release cells K562 were further constructed using the method of step (1) of this embodiment. Furthermore, in step (2) of this embodiment, releasing cells K562 and receptor cells Jurkat were co-cultured for 48 hours to observe whether contact between suspension cells can activate the SynNotch system. Simultaneously, releasing cells HT1080 and receptor cells Jurkat were co-cultured for 48 hours to observe whether contact between suspension cells and wall-attached cells can activate the SynNotch system. As shown in Figures 4C and 4D, both XENTR-SynNotch and MOUSE-SynNotch are effectively activated by contact between suspension receptor cells and suspension releasing cells, and by contact between suspension receiving cells and wall-attached releasing cells. In addition, the leak activation efficiency of XENTR-SynNotch is much lower than that of MOUSE-SynNotch, while the effective activation efficiency is slightly higher than that of MOUSE-SynNotch.

[0145] Therefore, the SynNotch system, which includes the transmembrane domain of the Notch receptor protein derived from the tropical frog (Xenopus tropicalis) as designed in this invention, has a stronger activation efficiency than the SynNotch receptor system, which includes the transmembrane domain of the original mouse-derived Notch receptor protein.

[0146] Example 3: Activation of Jurkat T cells by target cancer cells using a SynNotch-CAR gate-regulated chimeric antigen receptor.

[0147] Currently, T cells expressing chimeric antigen receptors (CARs) are effective in treating certain B-cell cancers. However, one of the main problems with CAR-T cell cancer immunotherapy is off-target effects, where therapeutic CAR-T cells destroy normal tissue, leading to serious side effects and even death. A possible way to mitigate this problem is to give therapeutic CAR-T cells more precise tumor recognition targets. Increasing the tumor antigen recognition by one and expressing a CAR to provide a more accurate T cell response is an effective method. To achieve this, it is hypothesized that SynNotch synthetic receptors can be used in the design of therapeutic CAR-T cells. Specifically, they first bind to tumor-specific cell surface antigens to detect the tumor, and then induce CAR expression only on a second tumor-specific antigen within the tumor, thereby providing dual-antigen control of CAR-T cell activity and a tumor localization response.

[0148] Based on this, the inventors have demonstrated that the inclusion of Jurkat cells (abbreviated as SynNotch-CAR-Jurkat T cells) in combination with a SynNotch-CAR gate-controlled chimeric antigen receptor can be determined by performing corresponding experiments with Jurkat cells (T cell lineage), specifically including the following steps: (1) The SynNotch synthesizer 1 in Example 2 is subjected to slow viral packaging. Simultaneously, the slow viral packaging can recognize the CD19 CAR molecule and includes a UAS-minimal-CMV sequence before the 5' end of the nucleic acid encoding the CAR molecule. This sequence binds to the intracellular polypeptide produced after the SynNotch synthesizer is activated, and can induce the expression of the CAR molecule (directly synthesized and slow-packaged by Kim Yu-Ji Co., Ltd., the CAR molecule has an amino acid sequence as shown in SEQ ID NO:10 and a nucleotide sequence as shown in SEQ ID NO:11, and subsequently referred to as the inducible CAR molecule). The above two viruses are used to simultaneously infect Jurkat T cells, and Jurkat T cells expressing the above SynNotch synthesizer and the inducible CAR molecule are stably selected. (2) Using conventional methods in this field, we construct K562 cells of single-expression membrane-fixed GFP, single-expression membrane-fixed CD19 (the structure of which is shown in Figure 5, and which has the amino acid sequence shown in SEQ ID NO:12 and the nucleotide sequence shown in SEQ ID NO:13), and co-expression membrane-fixed GFP and CD19 as transmission cells. (3) The three types of K562 cells from step (2) above were co-incubated with Jurkat cells constructed in step (1), and after 24 hours, the expression of CD69 and the secretion of IL-2 and TNF-α in each group of Jurkat T cells were detected. The results are shown in Figure 6. CD69 expression and the secretion of IL-2 and TNF-α represent activation of Jurkat T cells. As shown in Figure 6, SynNotch-CAR-Jurkat T cells are activated when exposed 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 (abbreviated as SynNotch-CAR-NK cells) combined with SynNotch-CAR gate-controlled chimeric antigen receptors.

[0150] The SynNotch synthetic receptor 1 according to the present invention is common not only to T cells but also to other types of immune cells. The inventors have confirmed that it functions similarly in other immune cells by applying a SynNotch-CAR system similar to that described in Example 4 to NK cells, specifically including the following steps. (1) The SynNotch synthesis receptor (SynNotch synthesis receptor 1 or SynNotch synthesis receptor 2) in Example 2 is subjected to slow viral packaging. Simultaneously, the slow viral packaging is capable of recognizing the CD19 CAR molecule and includes a single-step UAS-minimal-CMV sequence at the 5' end of the nucleic acid sequence encoding the CAR molecule. This sequence binds to an intracellular polypeptide produced after the SynNotch synthesis receptor is activated, inducing the expression of the CAR molecule (directly synthesized and slow-packaged by Kim Yu-Ji Co., Ltd., with the CAR molecule having an amino acid sequence as shown in SEQ ID NO:10 and a nucleotide sequence as shown in SEQ ID NO:11). The two viruses described above simultaneously infect NK cells, and by selection, NK cells that stably express the SynNotch synthesis receptor and CAR molecule are obtained. Simultaneously, NK cells that stably express and can recognize the CD19 CAR molecule (i.e., CAR-NK) are set as the positive killing control group, and NK-EV (NK cells infected with a virus produced by a blank plasmid) are set as the negative killing control group. (2) Construct Huh7 cells with single-expression membrane-fixed GFP, single-expression membrane-fixed CD19 (see step (2) of Example 3 for specific sequences), and co-expression membrane-fixed GFP and CD19 as signaling cells. (3) Label the signaling 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 (1), the cells were collected after 48 hours, dead cells were labeled with PI dye, and the viability of the Huh7 cells in each group was analyzed and detected, specifically refer to Figure 7. The mortality rate of Huh7 cells represents the killing status of the NK cells against the signaling cells. As shown in Figure 7, when exposed to Huh7 cells simultaneously expressing two antigens (GFP and CD19), SynNotch-CAR-NK cells are significantly activated and exhibit a higher killing effect against Huh7 cells. Compared to the combination of SynNotch-CAR containing SynNotch synthesis receptor 2 (M-Syn-CAR), the killing efficiency of SynNotch-CAR containing SynNotch synthesis receptor 1 (X-Syn-CAR) is almost improved, and the NK cell killing efficiency of SynNotch-CAR containing SynNotch synthesis receptor 1 is not significantly different compared to NK cells that stably express CAR molecules capable of recognizing CD19 (CAR-NK).

[0151] In this specification, reference terms such as “one embodiment,” “several embodiments,” “example,” “specific example,” or “several examples” refer to specific features, structures, materials, or characteristics described in combination with such embodiments or examples, which are included in at least one embodiment or example of the present invention. In this specification, the above-mentioned exemplary descriptions do not necessarily apply to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described can be combined in an appropriate manner in any or more embodiments or examples. Notwithstanding the fact that they do not conflict with each other, those skilled in the art can combine and combine different embodiments or examples and features of different embodiments or examples described herein.

[0152] Although embodiments of the present invention have been presented and described, these embodiments are illustrative and should not be understood as limiting the present invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations are possible in the above embodiments within the scope of the present invention.

Claims

1. The use of isolated polypeptides in the preparation of a SynNotch synthetic receptor, wherein the isolated polypeptides have 100% identity with the transmembrane region of a Notch receptor protein derived from the tropical clawed frog (Xenopus tropicalis), and the amino acid sequence of the transmembrane region of the tropical clawed frog Notch receptor protein is shown in SEQ ID NO:

1.

2. It is a chimeric polypeptide, An extracellular region having the activity to bind to the first molecule, A transmembrane region whose N end is connected to the C end of the extracellular region, and The intracellular region comprises an N-end connected to the C-end of the transmembrane region, The transmembrane region comprises a separated polypeptide, which has 100% identity with the transmembrane region of the Notch receptor protein derived from the tropical clawed frog. The amino acid sequence of the transmembrane region of the Notch receptor protein derived from the aforementioned African clawed frog is shown in SEQ ID NO:

1. The first molecule is a chimeric polypeptide characterized by comprising at least one of a tumor antigen, a virus, a bacterium, an endotoxin, an antibody, a cell receptor, and a ligand for a cell receptor.

3. The chimeric polypeptide according to claim 2, characterized in that the tumor antigen comprises at least one of a tumor-associated antigen and a tumor-specific antigen.

4. The first molecule mentioned above 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, CD 79A, CD79B, CD80, CD86, CD94, CD95, CD133, CD134, CD140, CD152, CD154, CD158, CD178, CD181, CD1 82, 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, GD 2. The chimeric polypeptide according to claim 2, characterized by comprising at least one of IL-13, IL-13RA2, ROR1, MUC1, PSMA, MAGEA1, 4-1BB, 5T4, BAFF, CA242, CA-IX, MET, CCR4, CNTO888, FAP, MORAb-009, VEGF-A, VEGFR-1, and VEGFR-2.

5. The chimeric polypeptide according to claim 2, characterized in that the extracellular region comprises a first binding protein or a fragment thereof that binds to the first molecule.

6. The chimeric polypeptide according to claim 5, characterized in that the first binding protein or fragment thereof comprises at least one of an antibody or a functional fragment thereof, a receptor, a ligand for a receptor, and a cell adhesion molecule.

7. The chimeric polypeptide according to claim 5, characterized in that the first binding protein or fragment thereof is a monoclonal antibody or polyclonal antibody that binds to the first molecule.

8. The chimeric polypeptide according to claim 7, characterized in that 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.

9. The chimeric polypeptide according to claim 2, characterized in that the intracellular region comprises at least one of a transcription-activating protein, a transcription-inhibiting protein, a transcription factor, a site-specific nuclease, a recombinant enzyme, an activating immune receptor intracellular structural domain, and an inhibitory immune receptor intracellular structural domain.

10. The chimeric polypeptide according to claim 9, characterized in that 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.

11. The chimeric polypeptide according to claim 10, characterized in that the amino acid sequence of Gal4-VP64 is shown in SEQ ID NO:

2.

12. The amino acid sequences of the transmembrane and intracellular regions are shown in SEQ ID NO: 3, and / or The chimeric polypeptide according to claim 2, characterized in that the amino acid sequence of the chimeric polypeptide is shown in SEQ ID NO:

4.

13. A first nucleic acid molecule, wherein the first nucleic acid molecule encodes a chimeric polypeptide according to any one of claims 2 to 12. The first nucleic acid molecule is characterized in that the nucleic acid molecule is DNA.

14. A first expression vector characterized by having the first nucleic acid molecule described in claim 13.

15. The first expression vector according to claim 14, characterized in that the first expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage.

16. Recombinant cells, Recombinant cells characterized by having the first nucleic acid molecule described in claim 13.

17. The recombinant cells are obtained by introducing the first expression vector described in claim 14 into host cells. The recombinant cell according to claim 16, characterized in that the host cell comprises at least one of immune cells, neurons, primordial or progenitor cells, epithelial cells, endothelial cells, and stem cells.

18. The recombinant cell according to claim 17, characterized in that the host cell comprises at least one of T cells, B cells, mononuclear cells, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells, and γδT cells.

19. The aforementioned recombinant cells further, Recombinant cells according to claim 16, comprising a second nucleic acid molecule encoding a chimeric antigen receptor, an NK cell receptor, or a T cell receptor, or a second expression vector having the second nucleic acid molecule, or expressing the chimeric antigen receptor, NK cell receptor, or T cell receptor.

20. The recombinant cell according to claim 19, characterized in that the intracellular region of the chimeric polypeptide comprises a transcription-activating protein, the 5' end of a second nucleic acid molecule encoding the chimeric antigen receptor, NK cell receptor, or T cell receptor is connected to an inducibly expressed nucleic acid sequence, and the inducibly expressed nucleic acid sequence is used to bind to the transcription-activating protein.

21. A pharmaceutical composition comprising a chimeric polypeptide according to any one of claims 2 to 12 and a pharmaceutically acceptable adjuvant.

22. Use of a chimeric polypeptide according to any one of claims 2 to 12 in the preparation of a drug for the prevention and / or treatment of a disease, The aforementioned diseases include cancer or tumors, autoimmune diseases, and related diseases due to inflammation and cellular senescence.

23. A therapeutic agent for preventing and / or treating a disease comprising a chimeric polypeptide according to any one of claims 2 to 12, A therapeutic agent for preventing and / or treating diseases, characterized in that the aforementioned diseases include cancer or tumors, autoimmune diseases, inflammation, and related diseases due to cellular senescence.

24. A method for activating immune cells in vitro, A method for activating immune cells, comprising the step of making a first contact between the immune cells and a first molecule, wherein the immune cells express a chimeric polypeptide according to any one of claims 2 to 12.

25. The intracellular domain of the chimeric polypeptide comprises a transcription-activating protein, an immune cell-expressed chimeric antigen receptor, an NK cell receptor, or a T cell receptor, the chimeric antigen receptor, NK cell receptor, or T cell receptor comprising an antibody or a functional fragment thereof that binds to a predetermined antigen, the 5' end of a second nucleic acid molecule encoding the chimeric antigen receptor, NK cell receptor, or T cell receptor being ligated to an inducible expression nucleic acid sequence, the inducible expression nucleic acid sequence being used to bind to the transcription-activating protein. The aforementioned method, Following the first contact, the immune cells release the transcription-activating protein and express the chimeric antigen receptor, NK cell receptor, or T cell receptor, The method further includes the step of binding the chimeric antigen receptor, NK cell receptor, or T cell receptor of the immune cell to the predetermined antigen and activating the immune cell, The method according to 24, characterized in that the immune cells include at least one of T cells, B cells, mononuclear cells, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells, and γδT cells.

26. A method for tracking the contact between a first cell and a second cell in vitro, A step of making a second contact between the first cell and the second cell, wherein the first cell expresses a chimeric polypeptide and a reporter gene protein according to any one of claims 2 to 12, the intracellular domain of the chimeric polypeptide is a transcription-activating protein, the 5' end of a third nucleic acid molecule encoding the reporter gene protein is attached to an inducible expression nucleic acid sequence, the inducible expression nucleic acid sequence is used to bind to the transcription-activating protein, and the second cell expresses the first molecule; A method for tracking contact between a first cell and a second cell, comprising the 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.

27. The first or second cell comprises at least one of immune cells, neurons, primordial or progenitor cells, epithelial cells, endothelial cells, and stem cells. The tracking method according to claim 26, characterized in that the first cell comprises at least one of T cells, B cells, mononuclear cells, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells, and γδT cells.

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