Chimeric transformation receptor and application thereof

Abstract

The invention provides a chimeric transformation receptor and an application thereof. The chimeric antigen receptor comprises an extracellular region, and the extracellular region comprises a TIGIT extracellular region and a PD-1 extracellular region; a transmembrane region; the intracellular region comprises a co-stimulation structural domain and an intracellular signal transduction structural domain, and the co-stimulation structural domain comprises a truncated 2B4 intracellular region and a truncated DAP10 intracellular region; wherein the N end of the transmembrane region is connected with the C end of the extracellular region, and the N end of the intracellular region is connected with the C end of the transmembrane region; and the C end of the costimulatory factor structural domain is connected with the N end of the intracellular signal transduction structural domain. Immune cells expressing the chimeric antigen receptor are high in tumor killing activity, high in degranulation level and high in interferon gamma generation level, the treatment effect of the immune cells based on the chimeric antigen receptor is further improved, and the clinical value is high.

Description

[0001] This application claims priority to Chinese application No. 202410295571.6 filed on March 14, 2024. The entire contents of Chinese application No. 202410295571.6 are incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of biopharmaceuticals, specifically, the present invention relates to a chimeric switch receptor and its application, specifically to a CSR-immune cell and its application in the field of tumor treatment, and more specifically, the present invention relates to a chimeric switch receptor that can simultaneously recognize two antigen ligands and corresponding nucleic acid molecules, expression vectors, lentiviral vectors, transgenic immune cells, pharmaceutical compositions and their uses. Background Art

[0003] Chimeric switch receptor (CSR) is an artificially constructed recombinant receptor that contains an extracellular antigen recognition domain, a transmembrane region, and an intracellular signal transduction domain. Unlike the extracellular antigen recognition domain of the chimeric antigen receptor, which is composed of a single-chain antibody, the extracellular antigen recognition domain of the chimeric switch receptor is mainly composed of inhibitory surface receptors of immune cells. The chimeric switch receptor recognizes the ligands of the inhibitory receptor, thereby converting the original immunosuppressive signal into an immune activation signal, activating immune cells and triggering an immune response against target cells expressing inhibitory receptor ligands. Immune cell therapy based on chimeric switch receptors is an emerging direction in the field of tumor treatment.

[0004] Chimeric converting receptor immune cell therapy has good application prospects in tumor treatment, but the efficacy still needs to be further improved, especially the intracellular region containing the co-stimulatory domain and the intracellular signaling domain, which has an important impact on the conversion of immunosuppressive signals into immune activation signals, the activation of immune cells, and the exertion of CSR functions. Summary of the Invention

[0005] The present invention aims to address, at least to some extent, at least one of the technical problems existing in the prior art. To this end, the present invention provides chimeric convert receptors and their applications. Immune cells expressing the chimeric convert receptors of the present invention exhibit strong tumor-killing activity, high levels of degranulation, and high levels of interferon-γ production. This invention further enhances the efficacy of immune cell therapy based on the chimeric convert receptor and has high clinical value.

[0006] It should be noted that the present invention is completed based on the following work of the inventors:

[0007] Tumors are heterogeneous. Although the ligands of the inhibitory receptor TIGIT and the inhibitory receptor PD-1 are highly expressed in tumor cells, they are often not expressed simultaneously even in the same tumor cell. For example, it is common for only a portion of tumor cells to express one ligand and another portion to express the other ligand. At the same time, tumor escape mechanisms can also lead to the loss of TIGIT or PD-1 ligands in tumor cells. Therefore, in the process of studying dual-chimeric converting receptors that can simultaneously recognize TIGIT and / or PD-1 ligands, the inventors found that the intracellular region containing the costimulatory domain and the intracellular signaling domain has a great influence on the therapeutic effect of immune cells expressing the above-mentioned dual-chimeric converting receptors.

[0008] Further experimental results showed that when the stimulatory domain includes a truncated 2B4 intracellular region and a DAP10 intracellular region, it can significantly enhance the tumor-killing activity of immune cells expressing the dual chimeric convert receptor, with high levels of degranulation and interferon-γ production, further improving the therapeutic effect of immune cells based on the chimeric convert receptor and having high clinical value.

[0009] Therefore, in the first aspect of the present invention, the present invention proposes a chimeric antigen receptor. According to an embodiment of the present invention, the chimeric antigen receptor includes: an extracellular region, the extracellular region includes the TIGIT extracellular region and the PD-1 extracellular region; a transmembrane region; an intracellular region, the intracellular region includes a costimulatory domain and an intracellular signaling domain, and the costimulatory domain includes a truncated 2B4 intracellular region and a DAP10 intracellular region; wherein the N-terminus of the transmembrane region is connected to the C-terminus of the extracellular region, and the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region; the C-terminus of the costimulatory factor domain is connected to the N-terminus of the intracellular signaling domain. According to an embodiment of the present invention, immune cells expressing the chimeric antigen receptor of the present invention have strong tumor killing activity, high degranulation levels, and high levels of interferon gamma production. The present invention further improves the therapeutic effect of immune cells based on chimeric conversion receptors and has high clinical value.

[0010] In a second aspect of the present invention, a nucleic acid molecule encoding the chimeric antigen receptor is provided. According to an embodiment of the present invention, the chimeric antigen receptor of the first aspect of the present invention can be expressed in immune cells carrying the nucleic acid molecule. The immune cells have strong tumor killing activity, good clinical efficacy, and high levels of degranulation and interferon gamma production when in contact with tumor cells expressing TIGIT and / or PD-1 ligands.

[0011] In a third aspect, the present invention provides an expression vector carrying the nucleic acid molecule of the second aspect of the present invention. Thus, the chimeric switch receptor of the first aspect of the present invention can be effectively expressed in cells using the constructed expression vector.

[0012] In a fourth aspect, the present invention provides a lentiviral vector, wherein the lentiviral vector carries a nucleotide sequence as shown in SEQ ID NO: 1 or 24. After the lentiviral vector of the fourth aspect of the present invention is introduced into a recipient cell, the recipient cell can express the chimeric switch receptor of the first aspect of the present invention.

[0013] In its fifth aspect, the present invention provides a transgenic immune cell expressing the chimeric switch receptor of the first aspect of the present invention, or carrying the nucleic acid molecule of the second aspect of the present invention, the expression vector of the third aspect of the present invention, or the lentiviral vector of the fourth aspect of the present invention. The resulting transgenic immune cell exhibits enhanced tumor-killing activity, increased degranulation levels, and interferon-γ production, and is useful for tumor treatment, exhibiting excellent clinical efficacy and high application value.

[0014] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the chimeric switch receptor of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the expression vector of the third aspect of the present invention, the lentiviral vector of the fourth aspect of the present invention, or the transgenic immune cell of the fifth aspect of the present invention. The resulting pharmaceutical composition can be further used for the prevention or treatment of tumor diseases.

[0015] In the seventh aspect of the present invention, the present invention proposes the use of the chimeric conversion receptor of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the expression vector of the third aspect of the present invention, the lentiviral vector of the fourth aspect of the present invention, the transgenic immune cell of the fifth aspect of the present invention, or the pharmaceutical composition of the sixth aspect of the present invention in the preparation of a drug, wherein the drug is used to prevent or treat TIGIT ligands and / or PD-1 ligand-mediated related diseases. The chimeric conversion receptor of the present invention and the corresponding nucleic acid molecule, expression vector, lentiviral vector, transgenic immune cell or pharmaceutical composition can be further prepared into a drug, which can be used clinically to prevent or treat TIGIT ligands and / or PD-1 ligand-mediated diseases.

[0016] Those skilled in the art will appreciate that the features and advantages described above for the chimeric switch receptor, nucleic acid molecule, expression vector, lentiviral vector, transgenic immune cell and pharmaceutical composition are also applicable to this use and will not be described in detail here.

[0017] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0019] Figure 1 Schematic diagram of the chimeric switch receptor structure of Example 1 of the present invention;

[0020] Figure 2 This is a graph showing the results of investigating the tumor cell cytotoxicity of NK cells expressing the chimeric switch receptor of Example 1 of the present invention, wherein "TP" represents NK cells expressing known dual chimeric switch receptors, "TP-newICD" represents NK cells expressing the chimeric switch receptor of Example 1 of the present invention, "CD155-SP K562" represents K562 cells overexpressing CD155, "PDL1-SP K562" represents K562 cells with CD112 and CD155 knocked out and overexpressing PDL1, and "DP K562" represents K562 cells overexpressing both PDL1 and CD155.

[0021] Figure 3 Figures 3A and 3B show the results of investigating TIGIT / PD1 ligand expression in solid tumor cell lines tested in Example 3 of the present invention, wherein (A), (B), and (C) depict the three tumor cell lines tested: human liver cancer cell line SNU423, human lung cancer cell line HCC827, and human esophageal cancer cell line TE-1;

[0022] Figure 4 This is a graph showing the results of investigating the solid tumor cell killing activity of NK cells expressing the chimeric switch receptor of Example 1 of the present invention;

[0023] Figure 5 Figures 4A and 4B show the results of investigating TIGIT / PD1 ligand expression in hematologic tumor cell lines tested in Example 4 of the present invention, wherein (A), (B), and (C) depict the three tumor cell lines tested, namely, the human multiple myeloma cell line LP-1, the human acute myeloid leukemia cell line HL60, and the non-Hodgkin's lymphoma cell line KARPAS-299;

[0024] Figure 6 This is a graph showing the results of an investigation into the killing activity of NK cells against hematological tumor cells expressing the chimeric switch receptor of Example 1 of the present invention;

[0025] Figure 7This figure shows the results of NK cell degranulation after NK cells expressing the chimeric switch receptor of Example 1 of the present invention are co-cultured with tumor cells, wherein "TP" represents NK cells expressing known dual chimeric switch receptors, "TP-newICD" represents NK cells expressing the chimeric switch receptor of Example 1 of the present invention, "155-SP K562" represents K562 cells overexpressing CD155, "PDL1-SP K562" represents K562 cells with CD112 and CD155 knocked out and overexpressing PDL1, and "DP K562" represents K562 cells overexpressing both PDL1 and CD155;

[0026] Figure 8 This figure shows the results of investigating the interferon-γ production level of NK cells after co-culture of NK cells expressing the chimeric switch receptor of Example 1 of the present invention with tumor cells, wherein "TP" represents NK cells expressing known dual chimeric switch receptors, "TP-newICD" represents NK cells expressing the chimeric switch receptor of Example 1 of the present invention, "155-SP K562" represents K562 cells overexpressing CD155, "PDL1-SP K562" represents K562 cells with CD112 and CD155 knocked out and overexpressing PDL1, and "DP K562" represents K562 cells overexpressing both PDL1 and CD155;

[0027] Figure 9 Schematic diagram of the structure of the known dual-chimeric switch receptor of Example 2 and Example 3 of the present invention. DETAILED DESCRIPTION

[0028] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0029] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0030] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0031] Terms and Definitions

[0032] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0033] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0034] In this article, the term "chimeric switch receptor" is equivalent to "CSR", equivalent to "Chimeric Switch Receptor", which is an artificially constructed recombinant receptor that contains an extracellular antigen recognition domain, a transmembrane region and an intracellular signal transduction domain. Unlike the extracellular antigen recognition domain of the chimeric antigen receptor, which is composed of a single-chain antibody, the extracellular antigen recognition domain of the chimeric switch receptor is mainly composed of inhibitory surface receptors of immune cells. In combination with the context, in this article, the term "chimeric switch receptor" is equivalent to "double chimeric switch receptor", "the chimeric switch receptor described in the present invention", "the above-mentioned chimeric switch receptor", and "the chimeric switch receptor of the first aspect of the present invention".

[0035] In this article, the term "immune cell expressing a chimeric conversion receptor" is equivalent to "CSR-immune cell", which is a genetically modified immune cell that expresses a specific chimeric conversion receptor and is further used for the prevention or treatment of a disease. In some specific cases, including but not limited to, it can be a CSR-T cell, or it can be a CSR-NK cell, CSR-NKT cell, CSR-γδT cell, CSR-macrophage, CSR-peripheral blood NK cell, CSR-umbilical cord blood NK cell or CSR-iPSC. In some cases, combined with the context, it is equivalent to "chimeric conversion receptor gene-modified immune cell technology" and "chimeric conversion receptor gene-modified immune cell".

[0036] In this article, the term "single-chain antibody" is equivalent to "single chain Fv" and "scFv", which is composed of the variable region of the immunoglobulin heavy chain (V H ) and light chain variable region (V L ) Small molecule antibodies connected by connecting peptides. The connecting peptides used to prepare scFv must be flexible enough to ensure V H and V L It can fold freely, so that the antibody binding region has the correct configuration.

[0037] In this article, the term "(G4S) n" is equivalent to "(Gly4Ser)n", which means 4 glycines and 1 serine are repeated n times. It is a widely used type of linker peptide that can be located at V H C-terminal and V L N terminal, can also be located at V L C-terminal and V H Between the N-termini, (G4S)3 is commonly used. Glycine, the amino acid with the smallest molecular weight and shortest side chain, increases side chain flexibility, while serine, the most hydrophilic amino acid, increases the hydrophilicity of the linker. Therefore, (G4S)3 exhibits excellent stability and activity. Linkers of varying lengths and sequences can be designed to construct scFvs with diverse biological functions.

[0038] In this article, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the inserted nucleic acid molecule is transferred into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for expression of the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell containing the vector.

[0039] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and thoroughly combining the active compound with a liquid carrier, a solid carrier, or both.

[0040] As used herein, the term "pharmaceutically acceptable excipient" includes any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the particular intended dosage form. Except to the extent that any conventional excipient is incompatible with the chimeric switch receptor, nucleic acid molecule, expression vector, lentiviral vector, or transgenic immune cell of the present invention, such as by producing any adverse biological effect or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present invention.

[0041] As used herein, the term "administer" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The chimeric switch receptor, nucleic acid molecule, expression vector, lentiviral vector, or transgenic immune cell or pharmaceutical composition of the present invention can be administered by any common route, as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, and the like, but the present invention is not limited to these exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous injection.

[0042] As used herein, the term "treatment" is used to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventative in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any administration of a drug or transgenic immune cell to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit the individual's disease, including but not limited to administering a drug containing cells containing a chimeric conversion receptor as described herein to an individual in need.

[0043] As used herein, "carbon terminus" and "C-terminus" are synonymous; "nitrogen terminus" and "N-terminus" are synonymous.

[0044] The present invention provides a chimeric switch receptor and corresponding nucleic acid molecule, expression vector, lentiviral vector, transgenic immune cell, pharmaceutical composition and pharmaceutical use thereof, which are described in detail below.

[0045] Chimeric antigen receptor

[0046] The present invention provides a chimeric antigen receptor. According to an embodiment of the present invention, the chimeric antigen receptor includes: an extracellular region, the extracellular region includes a TIGIT extracellular region and a PD-1 extracellular region; a transmembrane region; an intracellular region, the intracellular region includes a costimulatory domain and an intracellular signaling domain, the costimulatory domain includes a truncated 2B4 intracellular region and a DAP10 intracellular region; wherein the N-terminus of the transmembrane region is connected to the C-terminus of the extracellular region, and the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region; the C-terminus of the costimulatory factor domain is connected to the N-terminus of the intracellular signaling domain. According to an embodiment of the present invention, immune cells expressing the chimeric antigen receptor of the present invention have strong tumor killing activity, high degranulation levels, and high levels of interferon γ production. The present invention further improves the therapeutic effect of immune cells based on chimeric conversion receptors and has high clinical value.

[0047] Unexpectedly, with the same extracellular and transmembrane regions, the costimulatory domain includes a dual chimeric converting receptor targeting TIGIT ligand and PD-1 ligand, which includes a truncated 2B4 intracellular region and a DAP10 intracellular region. It has the ability to enhance the tumor cell killing activity of immune cells, as well as the activity of immune cells in activating NK cell degranulation and producing IFN-γ.

[0048] According to an embodiment of the present invention, the intracellular signaling domain is the intracellular segment of the CD3ζ molecule.

[0049] According to an embodiment of the present invention, the intracellular segment of the CD3ζ molecule has an amino acid sequence as shown in SEQ ID NO: 16.

[0050] According to an embodiment of the present invention, the transmembrane region includes the transmembrane segment of the CD8a molecule.

[0051] According to an embodiment of the present invention, the transmembrane segment of the CD8a molecule has an amino acid sequence as shown in SEQ ID NO: 13.

[0052] According to an embodiment of the present invention, the truncated 2B4 intracellular region has an amino acid sequence as shown in SEQ ID NO:14.

[0053] According to an embodiment of the present invention, the DAP10 intracellular region has the amino acid sequence shown in SEQ ID NO: 15.

[0054] According to an embodiment of the present invention, the extracellular region further includes a connecting peptide; the C-terminus of the TIGIT extracellular region is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the PD-1 extracellular region; or

[0055] According to an embodiment of the present invention, the C-terminus of the PD-1 extracellular region is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the TIGIT extracellular region.

[0056] According to an embodiment of the present invention, the connecting peptide is selected from at least one of (G4S)n, ESGRSGGGGSGGGGS, EGKSSGSGSESKST, EGKSSGSGSESKSTQ, GSTSGSGKSSEGKG, KESGSVSSEQLAQFRSLD, ESGSVSSEELAFRSLD, and (EA3K)n, where n is a non-zero integer.

[0057] According to an embodiment of the present invention, the connecting peptide is selected from (G4S) n , n is any integer between 2 and 6.

[0058] According to an embodiment of the present invention, the connecting peptide is (G4S)4.

[0059] According to an embodiment of the present invention, the TIGIT extracellular region has an amino acid sequence as shown in SEQ ID NO: 11.

[0060] According to an embodiment of the present invention, the PD-1 extracellular region has an amino acid sequence as shown in SEQ ID NO: 12.

[0061] According to an embodiment of the present invention, the chimeric switch receptor has an amino acid sequence as shown in SEQ ID NO: 9 or 25. The inventors obtained the preferred chimeric switch receptor through a large number of screening experiments.

[0062] Nucleic acid molecules

[0063] The present invention provides a nucleic acid molecule encoding the chimeric antigen receptor described above. According to embodiments of the present invention, immune cells carrying the nucleic acid molecule can express the chimeric antigen receptor. These immune cells exhibit strong tumor killing activity and good clinical efficacy. Upon contact with tumor cells expressing TIGIT and / or PD-1 ligands, they exhibit high levels of degranulation and interferon gamma production.

[0064] According to an embodiment of the present invention, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:8.

[0065] According to an embodiment of the present invention, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:5.

[0066] According to an embodiment of the present invention, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 6.

[0067] According to an embodiment of the present invention, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:7.

[0068] According to an embodiment of the present invention, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 3.

[0069] According to an embodiment of the present invention, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:4.

[0070] According to an embodiment of the present invention, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 24. After a large number of screening experiments, the inventors obtained a preferred nucleotide sequence encoding the aforementioned preferred chimeric switch receptor.

[0071] Thus, the expression level and / or expression efficiency of the aforementioned chimeric antigen receptor in immune cells is further improved.

[0072] It should be noted that, for nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases herein, the other strand complementary thereto is also disclosed. In addition, the molecular sequences in the present invention include DNA or RNA forms, and disclosure of one of them implies that the other is also disclosed.

[0073] expression vector

[0074] The present invention provides an expression vector. The expression vector carries the aforementioned nucleic acid molecule. Thus, the constructed expression vector can express the chimeric antigen receptor of the present invention in a recipient cell.

[0075] When the nucleic acid molecule is linked to an expression vector, the nucleic acid molecule can be directly or indirectly linked to the control elements on the expression vector, as long as these control elements are capable of controlling the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself, or they can be exogenous, that is, not derived from the vector itself. Of course, it is sufficient that the nucleic acid molecule is operably linked to the control elements. According to an embodiment of the present invention, the expression vector is a non-pathogenic viral vector.

[0076] As used herein, the term "operably linked" refers to the attachment of an exogenous gene to a vector so that control elements within the vector, such as transcriptional and translational control sequences, can function as intended to regulate the transcription and translation of the exogenous gene. Commonly used vectors include viral vectors, plasmids, and bacteriophages. Expression vectors according to certain embodiments of the present invention, upon introduction into appropriate recipient cells, can effectively express the aforementioned nucleic acid molecules under the mediation of a regulatory system, thereby enabling the in vitro production of large quantities of the protein encoded by the nucleic acid molecules.

[0077] According to an embodiment of the present invention, the non-pathogenic virus is selected from one of related viruses such as retrovirus, lentivirus, adenovirus and adeno-associated virus.

[0078] According to an embodiment of the present invention, the non-pathogenic virus is a lentivirus.

[0079] Lentiviral vectors

[0080] The present invention provides a lentiviral vector. The lentiviral vector carries a nucleotide sequence as shown in SEQ ID NO: 1 or 24. Thus, after the lentiviral vector is introduced into recipient cells, the chimeric antigen receptor of the present invention can be expressed in immune cells.

[0081] cell

[0082] The present invention provides a transgenic immune cell. The transgenic immune cell expresses the aforementioned chimeric antigen receptor; or carries the aforementioned nucleic acid molecule, expression vector, or lentiviral vector. The resulting transgenic immune cell exhibits significantly enhanced tumor cell-killing activity, effectively reducing its loss rate during tumor treatment.

[0083] According to an embodiment of the present invention, the genetically modified immune cells are selected from at least one of T cells, NK cells, macrophages, and hematopoietic stem cells.

[0084] According to some optional embodiments of the present invention, the T cells are selected from at least one of NK cells, NKT cells, and γδT cells, and the NK cells are selected from at least one of peripheral blood NK cells, umbilical cord blood NK cells, and NK-92 cells.

[0085] In an alternative embodiment of the present invention, the T cells, NK cells, macrophages, or hematopoietic stem cells are derived from iPSCs or differentiated from embryonic stem cells. It should be noted that when the stem cells described herein are human embryonic stem cells, they are stem cells isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development.

[0086] The chimeric antigen receptor of the present invention can be transduced into immune cells such as T, NK, NKT, γδT, macrophages, etc. through an expression vector (lentiviral vector or retroviral vector or non-viral vector system) and expressed on the surface of these immune cells.

[0087] According to an embodiment of the present invention, the transgenic immune cells of the present invention have better clinical efficacy and safety than immune cells modified with chimeric antigen receptor genes of monoclonal antibody antigen recognition domains administered sequentially or simultaneously under equivalent dosage and the same administration method.

[0088] Pharmaceutical composition

[0089] The present invention provides a pharmaceutical composition. The pharmaceutical composition comprises the aforementioned chimeric antigen receptor, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned lentiviral vector, or the aforementioned transgenic immune cell. The resulting pharmaceutical composition is further used for the treatment of tumors.

[0090] According to an embodiment of the present invention, the pharmaceutical composition further comprises: a pharmaceutically acceptable excipient.

[0091] Those skilled in the art will appreciate that the features and advantages described above for the chimeric antigen receptor, nucleic acid molecule, expression vector, lentiviral vector, and transgenic immune cells are also applicable to the pharmaceutical composition and will not be described in detail here.

[0092] use

[0093] The present invention provides a use of the above-mentioned chimeric antigen receptor, the above-mentioned nucleic acid molecule, the above-mentioned expression vector, the above-mentioned lentiviral vector, the above-mentioned transgenic immune cell or the above-mentioned pharmaceutical composition in the preparation of a drug, which is used to prevent or treat related diseases mediated by TIGIT ligands and / or PD-1 ligands.

[0094] According to an embodiment of the present invention, the disease is a tumor, and the tumor cells express a ligand of TIGIT and / or a ligand of PD-1.

[0095] According to an embodiment of the present invention, the tumor includes: a solid tumor or a hematological tumor.

[0096] According to an embodiment of the present invention, the solid tumor includes at least one of: pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, kidney cancer, breast cancer, prostate cancer, thyroid cancer, nasopharyngeal cancer, oral cancer, sarcoma, melanoma, and skin squamous cell carcinoma.

[0097] According to an embodiment of the present invention, the blood tumor includes: at least one of acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome and myeloproliferative tumors in blood cells and hematopoietic system.

[0098] Methods for preventing or treating tumors

[0099] The present invention provides a method for treating and / or preventing immune system diseases. According to an embodiment of the present invention, the method comprises: administering a pharmaceutically acceptable amount of the above-mentioned transgenic immune cells or the above-mentioned pharmaceutical composition to a subject.

[0100] The effective amount of the transgenic immune cells and pharmaceutical compositions described herein may vary depending on the mode of administration and the severity of the disease to be treated. The preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through 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 being treated, the patient's weight, the patient's immune status, and the route of administration. For example, if the urgency of the treatment is demanded, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0101] The sequences involved in the present invention are detailed in Table 1.

[0102] Table 1: Nucleotide / amino acid sequence description

[0103]

[0104]

[0105]

[0106]

[0107]

[0108] Below in conjunction with embodiment, the scheme of the present invention will be explained.It will be appreciated by those skilled in the art that the following examples are merely for illustration of the present invention and should not be considered as limiting the scope of the invention.The flow cytometer used in the embodiment is Beckman Cytoflex (purchased from Beckman Coulter), and flow cytometry data are analyzed using Cytexpert software (Beckman Coulter).Unrepresented specific techniques or conditions in the embodiment are described in accordance with the technology or conditions or according to product specifications.Reagents used or instruments not specified by manufacturer are conventional products that can be obtained commercially.

[0109] In the following examples, the methods for producing lentivirus and transducing human NK cells are as follows:

[0110] Replication-defective lentiviral vectors were prepared and collected by centrifugation for transduction of human NK cells. The following briefly describes the experimental procedures for lentiviral vector preparation, collection, and concentration: 293T cells were plated in a 150 cm² cell culture dish and transfected with plasmids using Lipofectamine 3000 (Thermo Fisher, Waltham, USA) according to the manufacturer's instructions. 47.37 μg of the lentiviral transgene plasmid, 30.8 μg of the psPAX2 plasmid, 16.58 μg of the pMD2.G plasmid, 189.48 μl of P3000, and 118.43 μl of Lipofectamine 3000 were added to each dish. After 24 hours, the supernatant was collected and centrifuged at 250g for 5 minutes (Hunan Kecheng L4-5K centrifuge) to remove the precipitate. The supernatant was then mixed with 1 / 4 volume of PEG-IT (purchased from Systems Biosciences, Palo Alto, USA) and incubated overnight at 4°C. The next day, the supernatant was centrifuged at 1500g for 30 minutes. Finally, the viral vector pellet was resuspended in 0.3 mL of serum-free DMEM medium.

[0111] Peripheral blood lymphocytes were isolated from the peripheral blood of an anonymous healthy volunteer donor by density gradient centrifugation using human peripheral blood lymphocyte separation medium (purchased from Dakoway, Shenzhen, China), and human primary NK cells were enriched using a human NK cell enrichment kit (purchased from Miltenyi, Bergisch Gladbach, Germany). Human NK cells (or human YTSNK cell line) were cultured in RPMI-1640 complete medium and stimulated with a final concentration of 1000 U / mL human IL-2 and a final concentration of 20 ng / mL human IL-21. After 48 hours of activation, 0.5 × 10 cells were plated per well in a 24-well plate. 6 NK cells were cultured in a volume of 0.3 mL of complete medium containing 100 U / mL 1640. 0.3 mL of the resuspended viral supernatant and Polybrene (8 μg / mL) were added to each well. After 12 hours, 0.45 mL of the culture supernatant was aspirated and 0.85 mL of complete medium containing 100 U / mL 1640 was added. The cells were cultured for another 3 days before use for functional analysis and subsequent experiments.

[0112] In the following examples, the cytotoxic activity of dual chimeric switch receptor NK cells was assessed using the in vitro Cell Trace CFSE-7AAD assay. The specific method is as follows:

[0113] The target test cells were labeled with a PBS solution containing a final concentration of 5 μM CFSE at 37°C for 15 minutes. After labeling, the cells were rinsed with RPMI 1640 medium containing 10% fetal calf serum (FCS). After rinsing, the cells were resuspended in the same medium at a concentration of 1×10 5 / mL. After transduction, NK cells were added to the target test cell suspension at different effector:target cell ratios (E:T), and the cells were seeded into 96-well round-bottom plates with a total volume of 200 μl per well. The cells were cultured in a 37°C incubator for 4 hours. After 4 hours, the entire cell suspension was aspirated from each well, and 3 μl of 7AAD solution was added to each well. After being placed in the dark for 1 minute, the proportion of 7AAD-positive cells in CFSE-positive cells was detected by flow cytometry using a Beckman Cytoflex (purchased from Beckman Coulter). This is the proportion of target cell death, which reflects the level of NK cell cytotoxic activity.

[0114] In the following examples, the ability of dual chimeric switch receptor NK cells to degranulate and produce interferon-γ was evaluated by co-culturing NK cells with tumor cells. The specific method is as follows:

[0115] Target test cells and transduced NK cells were seeded into 96-well round-bottom plates at an effector-to-target ratio (E:T) of 1:3. Each well contained a total volume of 200 μl, and 2 μl of anti-human CD107a fluorescent antibody was added. The cells were incubated at 37°C for 3 hours. After 3 hours, the entire cell suspension was aspirated from each well and labeled with anti-human CD56 fluorescent antibody. After incubation in the dark for 15 minutes, the cells were washed and fixed and permeabilized using fixative and transmembrane buffer (purchased from Biolegend, San Diego, USA) according to the manufacturer's instructions. The cells were then labeled with anti-human interferon-γ fluorescent antibody. After washing, the proportion of CD107a and interferon-γ-positive cells among CD56-positive NK cells was measured using a Beckman Cytoflex flow cytometer (purchased from Beckman Coulter). The proportion of CD107a-positive cells, representing the level of NK cell degranulation, was determined.

[0116] It should be noted that the "plasmid" and "vector" described in the following embodiments have the same meaning and can be used interchangeably.

[0117] Example 1: Construction of a vector expressing a dual chimeric switch receptor

[0118] In this example, a vector expressing a dual chimeric switch receptor was constructed as follows:

[0119] The sequences encoding human CD8α signal peptide (amino acid sequence as shown in SEQ ID NO: 10, nucleotide sequence as shown in SEQ ID NO: 2), human TIGIT extracellular domain sequence (amino acid sequence as shown in SEQ ID NO: 11, nucleotide sequence as shown in SEQ ID NO: 3), human PD-1 extracellular domain sequence (amino acid sequence as shown in SEQ ID NO: 12, nucleotide sequence as shown in SEQ ID NO: 4), human CD8α transmembrane domain sequence (amino acid sequence as shown in SEQ ID NO: 13, nucleotide sequence as shown in SEQ ID NO: 5), human truncated 2B4 intracellular domain sequence (amino acid sequence as shown in SEQ ID NO: 14, nucleotide sequence as shown in SEQ ID NO: 6), human DAP10 intracellular domain sequence (amino acid sequence as shown in SEQ ID NO: 15, nucleotide sequence as shown in SEQ ID NO: 7), and human T cell receptor CD3ζ intracellular segment sequence (amino acid sequence as shown in SEQ ID NO: 16, nucleotide sequence as shown in SEQ ID NO: 17). NO: 8) was cloned into a lentiviral vector containing an EF-1α promoter, and a lentiviral vector expressing a dual chimeric conversion receptor was obtained through enzyme digestion, ligation, screening, and amplification of the target plasmid.

[0120] Among them, the dual chimeric converting receptor is, from N-terminus to C-terminus, the sequence of the human TIGIT extracellular region (TIGIT ECD), the sequence of the human PD-1 extracellular region (PD-1ECD), the sequence of the human CD8α transmembrane region (CD8αTM), the human truncated 2B4 intracellular region sequence (t2B4), the human DAP10 intracellular region sequence (DAP10) and the human CD3ζ molecule intracellular segment sequence (CD3ζ).

[0121] The schematic diagram of the dual chimeric switch receptor structure of this embodiment is shown in Figure 1 , the specific sequence information is shown in Reference Table 1. Figure 1 In the present invention, "CD3Z" is synonymous with "CD3ζ".

[0122] Example 2: Tumor cell killing activity of NK cells expressing dual chimeric switch receptors

[0123] In this example, the inventors further investigated the killing effect of NK cells expressing the dual chimeric switch receptor of Example 1 on tumor cells expressing TIGIT and PD-1 ligands. The specific method is as follows:

[0124] Replication-defective lentiviral vectors were prepared and collected by centrifugation for transduction of human NK cells: 293T cells were plated in 150 cm2 cell culture dishes and transfected with plasmids using Lipofectamine 3000 (Thermo Fisher, Waltham, USA) according to the manufacturer's instructions. The cells were divided into four groups, each differing only in the lentiviral transgene plasmid: 47.37 μg of lentiviral transgene plasmids (Ctrl, TIGIT-NKR, PD1-NKR, TIGIT-PD1-NKR, and PD1-TIGIT-NKR), 30.8 μg of psPAX2 plasmid, 16.58 μg of pMD2.G plasmid, 189.48 μl of P3000, and 118.43 μl of Lipofectamine 3000 were added to each dish. After 24 hours, the supernatant was collected and centrifuged at 250g for 5 minutes (Hunan Kecheng L4-5K centrifuge) to remove the precipitate. The supernatant was then mixed with 1 / 4 volume of PEG-IT (purchased from Systems Biosciences, Palo Alto, USA) and incubated overnight at 4°C. The next day, the supernatant was centrifuged at 1500g for 30 minutes. Finally, the viral vector pellet was resuspended in 0.3 mL of serum-free DMEM medium.

[0125] Peripheral blood lymphocytes were isolated from the peripheral blood of an anonymous healthy volunteer donor by density gradient centrifugation using human peripheral blood lymphocyte separation medium (purchased from Dakoway, Shenzhen, China), and primary human NK cells were enriched using a human NK cell enrichment kit (purchased from Miltenyi, Bergisch Gladbach, Germany). Human NK cells were cultured in RPMI-1640 complete medium and stimulated with a final concentration of 1000 U / mL human IL-2 and a final concentration of 20 ng / mL human IL-21. After 48 hours of activation, 0.5 × 10 cells were plated per well in a 24-well plate. 6 NK cells were cultured in a volume of 0.3 mL complete medium containing 100 U / mL 1640. 0.3 mL of the four resuspended viral supernatants and Polybrene (8 μg / mL) were added to each well. After 12 hours, 0.45 mL of the culture supernatant was aspirated and 0.85 mL of complete medium containing 100 U / mL 1640 was added. Three to seven days after lentiviral vector transduction, transduced NK cells were harvested and used for in vitro CFSE-7AAD cytotoxicity assays.

[0126] There are three types of target cells tested: K562 overexpressing CD155 ("CD155-SP K562"), K562 knocking out CD112 and CD155 and overexpressing PDL1 ("PDL1-SP K562"), and K562 overexpressing both PDL1 and CD155 ("DP K562"). Each cell was labeled with a PBS solution containing a final concentration of 5 μM CFSE at 37°C for 15 minutes. After labeling, the cells were rinsed with RPMI medium containing 10% fetal calf serum (FCS). After rinsing, the cells were resuspended in the same medium at a concentration of 1×10 5 / mL. After transduction, NK cells were added to the target test cell suspension at a 1:1 effector / target cell ratio (E:T), and the cells were seeded into 96-well round-bottom plates in a total volume of 200 μl per well. The cells were incubated in a 37°C incubator for 4 hours. After 4 hours, the entire cell suspension was aspirated from each well, and 3 μl of 7AAD solution was added to each well. After incubation in the dark for 1 minute, the proportion of 7AAD-positive cells among CFSE-positive cells was measured using a Beckman Cytoflex flow cytometer (purchased from Beckman Coulter). This represents the proportion of target cell death and reflects the level of NK cell cytotoxic activity.

[0127] Among them, the known dual-chimeric switch receptor structures expressed by “TP” cells are as follows Figure 9 The amino acid sequence is shown in SEQ ID NO: 27, and the nucleotide sequence is shown in SEQ ID NO: 26.

[0128] The results are as follows Figure 2 shown.

[0129] The results showed that compared with NK92 cells expressing known dual-chimeric switch receptors ("TP"), NK cells expressing the dual-chimeric switch receptor of Example 1 ("TP-new ICD") had significantly improved killing rates for TIGIT ligand-positive, PD-1 ligand-negative tumor cells ("CD155-SP K562"), TIGIT ligand-negative, PD-1 ligand-positive tumor cells ("PDL1-SP K562"), and TIGIT / PD-1 ligand-double-positive tumor cells ("DP K562").

[0130] The above results indicate that the TIGIT-PD1 dual chimeric converting receptor of Example 1 1) can recognize tumor types with heterogeneous expression of TIGIT / PD1 ligands, and 2) compared with the TIGIT-PD1 dual chimeric converting receptor with known intracellular signal transduction domains, immune cells expressing the TIGIT-PD1 dual chimeric converting receptor of Example 1 have significantly enhanced tumor cell killing activity.

[0131] Referring to the method of Example 1, the inventors interchanged the sequence of the human TIGIT extracellular domain (amino acid sequence as shown in SEQ ID NO: 11, nucleotide sequence as shown in SEQ ID NO: 3) and the sequence of the human PD-1 extracellular domain (amino acid sequence as shown in SEQ ID NO: 12, nucleotide sequence as shown in SEQ ID NO: 4) in the order of connection, and obtained the PD1-TIGIT dual chimeric converting receptor as shown in SEQ ID NO: 25 and nucleotide sequence as shown in SEQ ID NO: 24. This PD1-TIGIT dual chimeric converting receptor was also observed to have significantly enhanced tumor cell killing activity.

[0132] The above results show that the dual chimeric converting receptor targeting TIGIT ligand and PD-1 ligand, whose costimulatory domain includes the truncated 2B4 intracellular region and the DAP10 intracellular region, has enhanced tumor cell killing activity.

[0133] Example 3: Cytotoxicity of NK cells expressing dual chimeric switch receptors against solid tumor cells

[0134] Referring to the method of Example 2, the inventors further investigated the killing activity of NK cells expressing the dual chimeric switch receptor of Example 1 against solid tumor cells.

[0135] Among them, there are 4 solid tumor cell lines tested, namely SNU423 (human liver cancer cell line), HCC827 (human lung cancer cell line), TE-1 (human esophageal cancer cell line), and HEY (human ovarian cancer cell line). Flow cytometry detection showed that these tumor cells expressed TIGIT ligands CD112, CD155 and / or PD1 ligands PDL1, PDL2 ( Figure 3 ). Each cell was labeled with a PBS solution containing a final concentration of 5 mM Cell Trace Violet at 37 degrees Celsius for 15 minutes. After labeling, the cells were rinsed with RPMI medium containing 10% fetal calf serum (FCS). After rinsing, the cells were resuspended in the same medium at a concentration of 1×10 5 / ml. After transduction, YTSNK cells were added to the target test cell suspension at an effector-to-target ratio (E:T) of 10:1, and the cells were seeded into 96-well round-bottom plates with a total volume of 200 μl per well. The cells were incubated in a 37°C incubator for 4 hours. After 4 hours, the entire cell suspension was aspirated from each well, and 3 μl of 7AAD solution was added to each well. After incubation in the dark for 1 minute, the proportion of 7AAD-positive cells among Cell Trace Violet-positive cells was measured using a Beckman Cytoflex flow cytometer (purchased from Beckman Coulter). This represents the proportion of target cell death and reflects the level of NK cell cytotoxic activity.

[0136] The results are as follows Figure 4 Among them, the known dual chimeric switch receptor structure expressed by “Ctrl” YTS NK cells is shown in Figure 9 The amino acid sequence is shown in SEQ ID NO: 27, and the nucleotide sequence is shown in SEQ ID NO: 26; the OR-gated chimeric switch receptor with optimized signal transduction domain expressed by "new ICD" YTSNK cells is the same as the dual chimeric switch receptor in Example 1.

[0137] The results showed that compared with YTS cells expressing OR-gated chimeric switch receptors containing known signal transduction domains, YTS cells expressing OR-gated chimeric switch receptors with optimized signal transduction domains in Example 1 had increased killing rates against several solid tumor cells, including SNU423 (human liver cancer cell line), HCC827 (human lung cancer cell line), TE-1 (human esophageal cancer cell line), and HEY (human ovarian cancer cell line).

[0138] Example 4: Cytotoxicity of NK cells expressing dual chimeric switch receptors against hematological tumor cells

[0139] Referring to the method of Example 2, the inventors further investigated the killing activity of NK cells expressing the dual chimeric switch receptor of Example 1 against hematological tumor cells.

[0140] Among them, there are three types of blood system tumor cells tested, namely LP-1 (multiple myeloma, MM), HL60 (acute myelogenous leukemia, AML), and KARPAS-299 (non-Hodgkin's lymphoma, NHL). Flow cytometry detection found that these tumor cells expressed TIGIT ligands CD112, CD155 and / or PD1 ligands PDL1, PDL2 ( Figure 5 ). Each cell was labeled with a PBS solution containing a final concentration of 5 mM CellTrace Violet at 37 degrees Celsius for 15 minutes. After labeling, the cells were rinsed with RPMI medium containing 10% fetal calf serum (FCS). After rinsing, the cells were resuspended in the same medium at a concentration of 1×10 5 / ml. After transduction, YTSNK cells were added to the target test cell suspension at an effector-to-target ratio (E:T) of 10:1, and the cells were seeded into 96-well round-bottom plates with a total volume of 200 μl per well. The cells were incubated in a 37°C incubator for 4 hours. After 4 hours, the entire cell suspension was aspirated from each well, and 3 μl of 7AAD solution was added to each well. After incubation in the dark for 1 minute, the proportion of 7AAD-positive cells among Cell Trace Violet-positive cells was measured using a Beckman Cytoflex flow cytometer (purchased from Beckman Coulter). This represents the proportion of target cell death and reflects the level of NK cell cytotoxic activity.

[0141] The results are as follows Figure 6 Among them, the known dual chimeric switch receptor structure expressed by “Ctrl” YTS NK cells is shown in Figure 9 The amino acid sequence is shown in SEQ ID NO: 27, and the nucleotide sequence is shown in SEQ ID NO: 26; the OR-gated chimeric switch receptor with optimized signal transduction domain expressed by "new ICD" YTSNK cells is the same as the dual chimeric switch receptor in Example 1.

[0142] The results showed that compared with YTS cells expressing OR-gated chimeric switch receptors containing known signal transduction domains, YTS cells expressing OR-gated chimeric switch receptors with optimized signal transduction domains in Example 1 had improved killing activity against several blood system tumor cells, including LP-1 (multiple myeloma, MM), HL60 (acute myelogenous leukemia, AML), and KARPAS-299 (non-Hodgkin's lymphoma, NHL).

[0143] The results of Examples 3 and 4 show that compared with OR-gated chimeric switch receptors containing known signal transduction domains, the OR-gated chimeric switch receptors containing novel intracellular signal transduction domains of the present invention can significantly enhance the killing activity of NK cells against solid tumor cells and hematological tumor cells.

[0144] Example 5: Effects of NK cells expressing dual chimeric switch receptors on degranulation and interferon-γ production in tumor cells expressing TIGIT and PD-1 ligands

[0145] In this example, the inventors further investigated the degranulation and interferon-γ production levels of NK cells expressing the dual chimeric switch receptor of Example 1 when stimulated by tumor cells expressing TIGIT and PD-1 ligands. The specific method is as follows:

[0146] Replication-defective lentiviral vectors were prepared and collected by centrifugation for transduction of human cells: 293T cells were plated in 150 cm2 cell culture dishes and transfected with plasmids using Lipofectamine 3000 (Thermo Fisher, Waltham, USA) according to the manufacturer's instructions. The cells were divided into four groups, each differing only in the lentiviral transgene plasmid: 47.37 μg of lentiviral transgene plasmids (Ctrl, TIGIT-NKR, PD1-NKR, TIGIT-PD1-NKR, and PD1-TIGIT-NKR), 30.8 μg of psPAX2 plasmid, 16.58 μg of pMD2.G plasmid, 189.48 μl of P3000, and 118.43 μl of Lipofectamine 3000 were added to each dish of cells. The supernatant was collected after 24 hours, and after centrifugation at 250g for 5 minutes (the centrifuge was Hunan Kecheng L4-5K), the precipitate was removed. The supernatant was mixed with 1 / 4 volume of PEG-IT (purchased from Systems Biosciences, Palo Alto, USA), placed overnight at 4°C, and centrifuged at 1500g for 30 minutes the next day. Finally, the viral vector pellet was resuspended in 0.3 mL of DMEM serum-free medium. Peripheral blood-derived lymphocytes were isolated from the peripheral blood of an anonymous healthy volunteer donor by density gradient centrifugation using human peripheral blood lymphocyte separation medium (purchased from Dakoway, Shenzhen, China), and primary human NK cells were enriched using a human NK cell enrichment kit (purchased from Miltenyi, Bergisch Gladbach, Germany). Human NK cells were cultured in RPMI-1640 complete medium and stimulated with human IL-2 at a final concentration of 1000 U / mL and human IL-21 at a final concentration of 20 ng / mL. After 48 hours of activation, 0.5×10 6 NK cells were cultured in a volume of 0.3 mL of complete medium containing 100 U / mL 1640. 0.3 mL of the resuspended viral supernatant and Polybrene (8 μg / mL) were added to each well. After 12 hours, 0.45 mL of the culture supernatant was aspirated and 0.85 mL of complete medium containing 100 U / mL 1640 was added. Three to seven days after lentiviral vector transduction, transduced NK cells were harvested and used for in vitro tumor cell stimulation experiments to detect degranulation and interferon-γ production.

[0147] Three target cell types were tested: K562 cells overexpressing CD155 ("155-SP K562"), K562 cells knocking out CD112 and CD155 and overexpressing PDL1 ("PDL1-SP K562"), and K562 cells overexpressing both PDL1 and CD155 ("DPK562"). These target cells and transduced NK cells were seeded into 96-well round-bottom plates at an effector:target ratio (E:T) of 1:4. Each well contained a total volume of 200 μl, and 2 μl of anti-human CD107a fluorescent antibody was added. The cells were incubated at 37°C for 3 hours. After 3 hours, all cell suspensions were aspirated from each well, labeled with anti-human CD56 fluorescent antibodies, incubated in the dark for 15 minutes, and then washed. The cells were fixed and transmembrane-transfected with fixative and transmembrane buffer (purchased from Biolegend, San Diego, USA) according to the instructions, and labeled with anti-human interferon-γ fluorescent antibodies. After washing, the proportion of CD107a and interferon-γ-positive cells in CD56-positive NK cells was detected by flow cytometry using Beckman Cytoflex (purchased from Beckman Coulter). The former is the level of NK cell degranulation.

[0148] Among them, the known dual-chimeric switch receptor structures expressed by “TP” cells are as follows Figure 9 The amino acid sequence is shown in SEQ ID NO: 27, and the nucleotide sequence is shown in SEQ ID NO: 26.

[0149] The results are as follows Figure 7 、 Figure 8 shown.

[0150] The results showed that compared with NK cells expressing known dual chimeric switch receptors ("TP"), the degranulation level and IFN-γ production level of NK cells expressing the dual chimeric switch receptor of Example 1 ("TP-new ICD") were significantly improved when they were co-cultured with TIGIT ligand-positive, PD-1 ligand-negative tumor cells ("155-SP K562"), TIGIT ligand-negative, PD-1 ligand-positive tumor cells ("PDL1-SP K562"), or TIGIT / PD-1 ligand-double-positive tumor cells ("DP K562").

[0151] The above results indicate that compared with the TIGIT-PD1 dual chimeric switch receptor with known intracellular signal transduction domains, the TIGIT-PD1 dual chimeric switch receptor of Example 1 can more strongly activate the NK cell degranulation level and IFN-γ production activity.

[0152] The PD1-TIGIT dual chimeric converting receptor having the amino acid sequence shown in SEQ ID NO: 25 and the novel intracellular costimulatory domain obtained by the method of Reference Example 1 was also observed to have enhanced activation of NK cell degranulation levels and IFN-γ production activity.

[0153] The above results show that the dual chimeric converting receptor targeting TIGIT ligand and PD-1 ligand, whose co-stimulatory domain includes the truncated 2B4 intracellular region and the DAP10 intracellular region, has enhanced the activity of activated NK cell degranulation and IFN-γ production.

[0154] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0155] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A chimeric switch receptor, characterized in that include: Extracellular region, comprising a TIGIT extracellular region and a PD-1 extracellular region; transmembrane region; an intracellular region comprising a costimulatory domain and an intracellular signaling domain, wherein the costimulatory domain comprises a truncated 2B4 intracellular region and a DAP10 intracellular region; wherein the N-terminus of the transmembrane region is connected to the C-terminus of the extracellular region, and the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region; The C-terminus of the costimulatory domain is connected to the N-terminus of the intracellular signaling domain.

2. The chimeric switch receptor according to claim 1, wherein The intracellular signal transduction domain is the intracellular segment of the CD3ζ molecule; Optionally, the intracellular segment of the CD3ζ molecule has the amino acid sequence shown in SEQ ID NO: 16; Optionally, the transmembrane region includes a transmembrane segment of a CD8a molecule; Optionally, the transmembrane segment of the CD8a molecule has the amino acid sequence shown in SEQ ID NO: 13; Optionally, the truncated 2B4 intracellular region has the amino acid sequence shown in SEQ ID NO: 14; Optionally, the DAP10 intracellular region has the amino acid sequence shown in SEQ ID NO:

15.

3. The chimeric switch receptor according to claim 1, wherein The extracellular region further includes a connecting peptide; the C-terminus of the TIGIT extracellular region is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the PD-1 extracellular region; or The C-terminus of the PD-1 extracellular region is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the TIGIT extracellular region; Optionally, the connecting peptide is selected from at least one of (G4S)n, ESGRSGGGGSGGGGS, EGKSSGSGSESKST, EGKSSGSGSESKSTQ, GSTSGSGKSSEGKG, KESGSVSSEQLAQFRSLD, ESGSVSSEELAFRSLD, and (EA3K)n, where n is an integer not equal to zero; Optionally, the connecting peptide is selected from (G4S) n , n is any integer between 2 and 6; Optionally, the connecting peptide is (G4S)4; Optionally, the TIGIT extracellular region has the amino acid sequence shown in SEQ ID NO: 11; Optionally, the PD-1 extracellular region has the amino acid sequence shown in SEQ ID NO:

12.

4. The chimeric switch receptor according to claim 1, wherein The chimeric switch receptor has the amino acid sequence shown in SEQ ID NO: 9 or 25.

5. A nucleic acid molecule, characterized in that Encoding the chimeric switch receptor according to any one of claims 1 to 4; Optionally, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 8; Optionally, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 5; Optionally, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 6; Optionally, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 7; Optionally, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 3; Optionally, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 4; Optionally, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

24.

6. An expression vector, characterized in that Carrying the nucleic acid molecule according to claim 5; Optionally, the expression vector is a non-pathogenic viral vector; Optionally, the non-pathogenic virus is selected from one of related viruses such as retrovirus, lentivirus, adenovirus and adeno-associated virus; Optionally, the non-pathogenic virus is a lentivirus.

7. A lentiviral vector, characterized in that Carrying the nucleotide sequence shown in SEQ ID NO: 1 or 24.

8. A transgenic immune cell, characterized in that: The transgenic immune cells include: Expressing the chimeric switch receptor according to any one of claims 1 to 4; or Carrying the nucleic acid molecule of claim 5, the expression vector of claim 6, or the lentiviral vector of claim 7; Optionally, the genetically modified immune cells are selected from at least one of T cells, NK cells, macrophages, and hematopoietic stem cells; Optionally, the T cells are selected from at least one of NK cells, NKT cells, and γδT cells, and the NK cells are selected from at least one of peripheral blood NK cells, umbilical cord blood NK cells, and NK-92 cells; Optionally, the T cells, NK cells, macrophages or hematopoietic stem cells are derived from iPSCs or differentiated from embryonic stem cells.

9. A pharmaceutical composition, characterized in that include: The chimeric switch receptor according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, the lentiviral vector according to claim 7, or the transgenic immune cell according to claim 8; Optionally, it further comprises: pharmaceutically acceptable excipients.

10. Use of the chimeric switch receptor according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, the lentiviral vector according to claim 7, the transgenic immune cell according to claim 8, or the pharmaceutical composition according to claim 9 in the preparation of a drug, characterized in that: The drug is used to prevent or treat diseases mediated by TIGIT ligands and / or PD-1 ligands; Optionally, the disease is a tumor, and the tumor cells express a ligand of TIGIT and / or a ligand of PD-1; Optionally, the tumor comprises: a solid tumor or a hematological tumor; Optionally, the solid tumor comprises at least one of pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, kidney cancer, breast cancer, prostate cancer, thyroid cancer, nasopharyngeal cancer, oral cancer, sarcoma, melanoma, and skin squamous cell carcinoma; Optionally, the blood tumor includes at least one of acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome and myeloproliferative neoplasms in blood cells and the hematopoietic system.

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