Novel chimeric antigen receptor modified immune cell and application thereof in preparation of drugs

Abstract

The invention belongs to the technical field of biology, and particularly relates to a novel chimeric antigen receptor modified immune cell and application of the novel chimeric antigen receptor modified immune cell in preparation of drugs, and the drugs can improve the anti-tumor cell factor secretion capacity of the immune cell; wherein the immune cell is modified with a chimeric antigen receptor, and the chimeric antigen receptor comprises an extracellular region, a transmembrane region and an intracellular region; the extracellular region can be combined with a tumor antigen; the transmembrane region is connected with the extracellular region; the intracellular region is connected with the transmembrane region; the intracellular region is selected from all amino acid sequences or partial amino acid sequences of at least one of the following proteins: 2B4, DAP10, CD3 zeta and SAP; the extracellular region is derived from an NK cell activation receptor; the tumor antigen is a tumor specific antigen or a tumor associated antigen. According to the application, the novel chimeric antigen receptor modified immune cell is applied to preparation of anti-tumor drugs, and by means of the innovatively designed chimeric antigen receptor, the anti-tumor cell factor secretion capacity of the immune cell is remarkably enhanced; according to the present invention, the CAR-immune cell immunotherapy kit has characteristics of high sensitivity and high efficiency, can accurately identify and efficiently attack a variety of NKG2D ligand positive tumor cells, can reduce the non-specific attack on normal cells, can significantly improve the safety and the effectiveness when the CAR-immune cell immunotherapy kit is used for treatment, and can provide a new idea for the CAR-immune cell therapy.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to novel chimeric antigen receptor-modified immune cells and their use in drug preparation. Background Technology

[0002] Immunotherapy primarily utilizes chimeric antigen receptor (CAR) modification of immune cells to enhance their ability to recognize and kill tumor cells. In recent years, chimeric antigen receptor T-cell (CAR-T) drugs have made significant progress in the treatment of hematological malignancies. These drugs achieve high specificity and efficient killing of tumor cells by fusing the antigen-binding ability of antibodies with the activation signals of T cells. Currently, CAR design mainly focuses on T cells, with insufficient optimization of other immune cells (such as NK cells), resulting in limited cytokine secretion capacity and difficulty in effectively treating solid tumors. Applying the CAR-T design concept directly to natural killer (NK) cells presents numerous challenges. This is because T cells and NK cells differ fundamentally in their functional characteristics and tumor attack mechanisms. CAR-T relies on the CD3ζ chain to activate T cells, while NK cell activation depends on a complex network of receptors and signaling molecules. Furthermore, NK cells possess a powerful tumor recognition system, NKR, on their surface. Simply replicating the CAR-T design logic ignores the unique biological advantages of NK cells, leading to suboptimal efficacy.

[0003] Therefore, developing a novel chimeric antigen receptor structure suitable for NK cells and applying it to the preparation of anti-tumor drugs is of great significance for overcoming the limitations of traditional CAR technology and improving the efficacy of tumor treatment. Summary of the Invention

[0004] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention provides novel chimeric antigen receptor-modified immune cells and their use in drug preparation. The purpose of this invention is the application of a novel chimeric antigen receptor-modified immune cell in the preparation of anti-tumor drugs. This immune cell, through an innovatively designed chimeric antigen receptor, significantly enhances the anti-tumor cytokine secretion capacity of immune cells (especially NK cells), and can accurately identify and efficiently attack various NKG2D ligand-positive tumor cells while reducing non-specific attacks on normal cells. This significantly improves the safety and efficacy of its application in treatment, providing new ideas and strategies for CAR-immunotherapy, and demonstrating extremely high application prospects and clinical value.

[0005] In a first aspect, the present invention provides the use of immune cells in the preparation of a drug. According to embodiments of the invention, the drug can enhance the anti-tumor cytokine secretion capacity of the immune cells; wherein the immune cells are modified with a chimeric antigen receptor, the chimeric antigen receptor comprising an extracellular region, a transmembrane region, and an intracellular region; the extracellular region is capable of binding to a tumor antigen; the transmembrane region is connected to the extracellular region; the intracellular region is connected to the transmembrane region; the intracellular region is selected from the complete or partial amino acid sequence of at least one of the following proteins: 2B4, DAP10, CD3ζ, and SAP; the extracellular region is derived from an NK cell activation receptor; the tumor antigen is a tumor-specific antigen or a tumor-associated antigen. According to the use of embodiments of the invention, the innovatively designed chimeric antigen receptor significantly enhances the anti-tumor cytokine secretion capacity of immune cells, and can accurately identify and efficiently attack various NKG2D ligand-positive tumor cells, reducing non-specific attacks on normal cells, significantly improving the safety and efficacy of its application in treatment, providing a new idea and strategy for CAR-immunotherapy, with broad application prospects.

[0006] According to embodiments of the present invention, the above-described uses may also have the following additional technical features:

[0007] According to embodiments of the present invention, the antitumor cytokines include one or more of interferon-γ, tumor necrosis factor-α, and interleukins.

[0008] According to embodiments of the present invention, the immune cells include one or more of the above-mentioned immune cells derived from T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells, γδT cells, and iPSCs.

[0009] According to an embodiment of the present invention, the immune cell is an NK cell.

[0010] According to an embodiment of the present invention, the intracellular region of the chimeric antigen receptor is selected from the complete or partial amino acid sequences of at least two of the following proteins: 2B4, DAP10, CD3ζ, and SAP.

[0011] According to an embodiment of the present invention, the structure of the chimeric antigen receptor is as follows: the extracellular region is the extracellular domain of an NK cell activation receptor or a part thereof, the transmembrane region is the transmembrane region of an NK cell activation receptor or a part thereof, the extracellular region and the transmembrane region originate from the same or different NK cell activation receptors, or the transmembrane region originates from a type I or type II transmembrane protein or a synthetically produced transmembrane protein with hydrophobic helices.

[0012] According to embodiments of the present invention, the extracellular region, transmembrane region, and intracellular region are directly connected or connected by connectors.

[0013] According to an embodiment of the present invention, the NK cell activation receptor is selected from one or more of NKG2D, DNAM-1, 2B4, NKp30, NKp46, NKp44, NKp80, NKG2C and NKG2E.

[0014] According to an embodiment of the present invention, the NK cell activation receptor is NKG2D.

[0015] According to an embodiment of the present invention, the segment after the extracellular region and the transmembrane region are connected constitutes the NKG2D protein.

[0016] According to embodiments of the present invention, the chimeric antigen receptor is selected from any of the following structures:

[0017] (a): [SAP]-[CD3ζ-ICD]-[2B4-ICD]-[NKG2D];

[0018] (b): [SAP]-[CD3ζ-ICD]-[DAP10-ICD]-[2B4-ICD]-[NKG2D];

[0019] (c): [CD3ζ-ICD]-[t2B4-ICD]-[NKG2D];

[0020] (d): [CD3ζ-ICD]-[DAP10-ICD]-[t2B4-ICD]-[NKG2D];

[0021] (e): [SAP]-[CD3ζ-ICD]-[t2B4-ICD]-[NKG2D];

[0022] In this context, "[]" represents a structural domain, and "]-[" represents a connection between structural domains.

[0023] The connections between the structural domains are either direct connections or connections via connectors;

[0024] CD3ζ-ICD, DAP10-ICD, and 2B4-ICD represent the intracellular activation domains of the CD3ζ, DAP10, and 2B4 proteins.

[0025] t2B4-ICD is a truncated portion of the intracellular activation domain of the 2B4 protein.

[0026] According to embodiments of the present invention, the amino acid sequence of CD3ζ-ICD is shown in SEQ ID NO: 1, the amino acid sequence of 2B4-ICD is shown in SEQ ID NO: 2, the amino acid sequence of t2B4-ICD is shown in SEQ ID NO: 3, the amino acid sequence of DAP10-ICD is shown in SEQ ID NO: 4, the amino acid sequence of SAP is shown in SEQ ID NO: 5, and the amino acid sequence of NKG2D is shown in SEQ ID NO: 6.

[0027] In a second aspect, the present invention provides a chimeric antigen receptor. According to embodiments of the invention, it comprises: an extracellular region capable of binding to a tumor antigen; a transmembrane region connected to the extracellular region; and an intracellular region connected to the transmembrane region; wherein the intracellular region is selected from the complete or partial amino acid sequence of at least one of the following proteins: 2B4, DAP10, CD3ζ, and SAP; the extracellular region is derived from an NK cell activation receptor; and the tumor antigen is a tumor-specific antigen or a tumor-associated antigen. The chimeric antigen receptor according to embodiments of the present invention integrates multiple key signal transduction elements (such as 2B4, DAP10, CD3ζ, and SAP) in its intracellular region, and its extracellular region is derived from an NK cell activation receptor (such as NKG2D), capable of specifically binding to tumor antigens.

[0028] According to embodiments of the present invention, the chimeric antigen receptor may further have the following additional technical features:

[0029] According to an embodiment of the present invention, the intracellular region of the chimeric antigen receptor is selected from the complete or partial amino acid sequences of at least two of the following proteins: 2B4, DAP10, CD3ζ, and SAP.

[0030] According to an embodiment of the present invention, the structure of the chimeric antigen receptor is as follows: the extracellular region is the extracellular domain of an NK cell activation receptor or a part thereof, the transmembrane region is the transmembrane region of an NK cell activation receptor or a part thereof, the extracellular region and the transmembrane region originate from the same or different NK cell activation receptors, or the transmembrane region originates from a type I or type II transmembrane protein or a synthetically produced transmembrane protein with hydrophobic helices.

[0031] According to embodiments of the present invention, the extracellular region, transmembrane region, and intracellular region are directly connected or connected by connectors.

[0032] According to an embodiment of the present invention, the NK cell activation receptor is selected from one or more of NKG2D, DNAM-1, 2B4, NKp30, NKp46, NKp44, NKp80, NKG2C and NKG2E.

[0033] According to an embodiment of the present invention, the NK cell activation receptor is NKG2D.

[0034] According to an embodiment of the present invention, the segment after the extracellular region and the transmembrane region are connected constitutes the NKG2D protein.

[0035] According to embodiments of the present invention, the chimeric antigen receptor is selected from any of the following structures:

[0036] (a): [SAP]-[CD3ζ-ICD]-[2B4-ICD]-[NKG2D];

[0037] (b): [SAP]-[CD3ζ-ICD]-[DAP10-ICD]-[2B4-ICD]-[NKG2D];

[0038] (c): [CD3ζ-ICD]-[t2B4-ICD]-[NKG2D];

[0039] (d): [CD3ζ-ICD]-[DAP10-ICD]-[t2B4-ICD]-[NKG2D];

[0040] (e): [SAP]-[CD3ζ-ICD]-[t2B4-ICD]-[NKG2D];

[0041] In this context, "[]" represents a structural domain, and "]-[" represents a connection between structural domains.

[0042] The connections between the structural domains are either direct connections or connections via connectors;

[0043] CD3ζ-ICD, DAP10-ICD, and 2B4-ICD represent the intracellular activation domains of the CD3ζ, DAP10, and 2B4 proteins.

[0044] t2B4-ICD is a truncated portion of the intracellular activation domain of the 2B4 protein.

[0045] According to embodiments of the present invention, the amino acid sequence of CD3ζ-ICD is shown in SEQ ID NO: 1, the amino acid sequence of 2B4-ICD is shown in SEQ ID NO: 2, the amino acid sequence of t2B4-ICD is shown in SEQ ID NO: 3, the amino acid sequence of DAP10-ICD is shown in SEQ ID NO: 4, the amino acid sequence of SAP is shown in SEQ ID NO: 5, and the amino acid sequence of NKG2D is shown in SEQ ID NO: 6.

[0046] In a third aspect, the present invention provides a nucleic acid molecule. According to embodiments of the invention, the nucleic acid molecule encodes the chimeric antigen receptor described in the second aspect. The nucleic acid molecule according to embodiments of the invention encodes the aforementioned chimeric antigen receptor, enabling the aforementioned chimeric antigen receptor to be expressed efficiently and stably in immune cells.

[0047] In a fourth aspect, the present invention provides an expression vector. According to an embodiment of the invention, the expression vector comprises the nucleic acid molecule described in the third aspect. The expression vector according to an embodiment of the invention integrates the nucleic acid molecule encoding the aforementioned chimeric antigen receptor into the expression vector, thereby enabling the chimeric antigen receptor gene to be introduced into immune cells efficiently and stably expressed in immune cells.

[0048] In a fifth aspect, the present invention provides a recombinant virus. According to an embodiment of the invention, the recombinant virus comprises the expression vector described in the fourth aspect, and the recombinant virus is capable of infecting immune cells. The recombinant virus according to an embodiment of the invention can efficiently infect immune cells, thereby achieving efficient and stable expression of the aforementioned chimeric antigen receptor in immune cells, providing a method for the preparation of modified cells such as CAR-NK cells or CAR-T cells.

[0049] In a sixth aspect, the present invention provides an immune cell. According to embodiments of the invention, the immune cell carries the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect, or is capable of expressing the chimeric antigen receptor described in the second aspect. The immune cell according to embodiments of the invention, by expressing the optimized chimeric antigen receptor described above, significantly enhances the anti-tumor cytokine secretion capacity of immune cells (especially NK cells).

[0050] According to embodiments of the present invention, the above-mentioned immune cells may further have the following additional technical features:

[0051] According to embodiments of the present invention, the immune cells include one or more of the above-mentioned immune cells derived from T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells, γδT cells, and iPSCs.

[0052] According to an embodiment of the present invention, the immune cell is an NK cell.

[0053] In a seventh aspect, the present invention provides a composition. According to embodiments of the invention, the composition comprises one or more of the following: the chimeric antigen receptor described in the second aspect; the nucleic acid molecule described in the third aspect; the expression vector described in the fourth aspect; and the immune cell described in the sixth aspect. The composition according to embodiments of the invention, by providing the aforementioned chimeric antigen receptor, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned immune cell, provides a new approach for the clinical application of CAR therapy.

[0054] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0055] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0056] Figure 1 This is a schematic diagram of NeoN-TCAD and a schematic diagram of NeoN-NeoCAD-1 to NeoCAD-5 combinations in Embodiment 1 of the present invention;

[0057] Figure 2 This is a flow cytometry analysis result of NKG2D expression in each group of NK cells in Example 2 of the present invention;

[0058] Figure 3 The following are flow cytometry results of the expression of NKG2D ligand MICA / B in different types of tumor cells in Example 3 of the present invention: (A) is the flow cytometry result of NCI-H716 colon cancer cells, (B) is the flow cytometry result of K562 leukemia cells, (C) is the flow cytometry result of MDA-MB-231 breast cancer cells, and (D) is the flow cytometry result of PLC / PRF / 5 hepatocellular carcinoma cells.

[0059] Figure 4 The following are the results of in vitro cytokine secretion function tests of different NK cells in Example 4 of the present invention: (A) is the result of in vitro cytokine IFN-γ secretion function tests of different NK cells, and (B) is the result of in vitro cytokine TNF-α secretion function tests of different NK cells.

[0060] Figure 5 The figures shown are the results of in vitro cytokine secretion function tests of different T cells (Jurkat cells) in Example 4 of the present invention. (A) shows the results of in vitro cytokine IFN-γ secretion function tests of different Jurkat cells, and (B) shows the results of in vitro cytokine TNF-α secretion function tests of different Jurkat cells. Detailed Implementation

[0061] 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 construed as limiting the present invention.

[0062] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0063] The endpoints and any values ​​of the ranges 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 endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0064] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0065] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0066] Terms and Definitions

[0067] In this article, the term "chimeric antigen receptor" is abbreviated as CAR. It is an artificial receptor molecule manufactured using genetic engineering techniques, which includes an extracellular region, an intracellular region, and a transmembrane region.

[0068] In this article, the term "NKG2D ligand positive" refers to the state of cell surface expression of NKG2D ligand. NKG2D ligand is a class of proteins that can be recognized by NKG2D receptors. It is mainly expressed on the surface of stressed cells (such as tumor cells or virus-infected cells) and is an important target for NK cells and some T cells to recognize and eliminate abnormal cells. It can provide a clear target for CAR-immune cells and help improve the specificity and effectiveness of treatment.

[0069] In this paper, the term "expression vector" refers to a specially designed nucleic acid molecular tool primarily used to achieve efficient transcription and translation of exogenous genes in host cells, thereby producing the desired protein or peptide. Expression vectors typically contain a strong promoter (such as CMV or EF1α) to drive efficient transcription, a multiple cloning site for inserting the target gene, a terminator to ensure proper termination of transcription, and selection marker genes (such as puromycin resistance genes) and reporter genes (such as GFP) for screening and monitoring gene expression. Furthermore, expression vectors may contain an origin of replication to ensure stable replication in the host cell, and optimized transcriptional and translational regulatory elements to improve expression efficiency. Expression vectors not only efficiently introduce exogenous genes into host cells but also achieve long-term stable gene expression in various cell types.

[0070] In this document, the term "treatment" refers to the use of drugs to achieve desired pharmacological and / or physiological effects. These effects may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in susceptible individuals who have not yet been diagnosed with the disease; (b) suppression of disease, such as inhibiting disease progression; or (c) alleviating disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any medication that administers a drug or bispecific antibody to an individual to treat, cure, alleviate, improve, reduce, or suppress the individual's disease, including but not limited to administering a drug containing the drugs described herein to an individual in need.

[0071] In this document, "immune cells" refers to cells derived from humans. More specifically, the immune cells in this invention can be derived from pluripotent stem cells through directed differentiation. Depending on the different differentiation conditions provided, these immune cells can be T cells, NK cells, and macrophages. The immune cells in this invention can also be derived from human hematopoietic stem cells. These hematopoietic stem cells can be isolated from peripheral blood, umbilical cord blood, placental blood, or differentiated from pluripotent stem cells; depending on the different differentiation conditions provided, these immune cells can be T cells, NK cells, and macrophages; the immune cells in this invention can also be isolated from human blood, bone marrow, lymph nodes or lymphoid organs, or specific tissues (including but not limited to tumor tissue, abdominal cavity, liver, lungs, and other organs, muscles, etc.). For example, cells of innate or adaptive immunity, such as bone marrow or lymphocytes, are typically T cells, NK cells, and macrophages; preferably, according to this invention, immune cells include NK cells and T cells; the immune cells of this invention can also be derived from immune cell progenitor cells, such as lymphocyte progenitor cells.

[0072] In this document, "composition" means a form in which the biological activity of the active ingredient is present and which does not contain any other ingredients that would have unacceptable toxicity to the subject to which the composition is applied.

[0073] use

[0074] This invention proposes the use of immune cells in drug preparation. According to embodiments of the invention, the drug can enhance the anti-tumor cytokine secretion capacity of the immune cells; wherein the immune cells are modified with a chimeric antigen receptor, the chimeric antigen receptor comprising an extracellular region, a transmembrane region, and an intracellular region; the extracellular region is capable of binding to tumor antigens; the transmembrane region is connected to the extracellular region; the intracellular region is connected to the transmembrane region; the intracellular region is selected from the complete or partial amino acid sequence of at least one of the following proteins: 2B4, DAP10, CD3ζ, and SAP; the extracellular region is derived from NK cell activation receptors; the tumor antigen is a tumor-specific antigen or a tumor-associated antigen. According to the use of embodiments of the invention, the innovatively designed chimeric antigen receptor significantly enhances the anti-tumor cytokine secretion capacity of immune cells, and can accurately identify and efficiently attack various NKG2D ligand-positive tumor cells, reducing non-specific attacks on normal cells, significantly improving the safety and efficacy of its application in treatment, providing a new idea and strategy for CAR-immunotherapy, with broad application prospects.

[0075] According to embodiments of the present invention, the antitumor cytokines include one or more of interferon-γ, tumor necrosis factor-α, and interleukins. These antitumor cytokines play a crucial role in immune cell-mediated antitumor responses; thereby, the drug significantly enhances the ability of immune cells to secrete antitumor cytokines, thereby increasing the activity of immune cells and their killing effect on tumor cells.

[0076] According to embodiments of the present invention, the immune cells include one or more of the following: T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells, γδT cells, and iPSC-derived immune cells. The chimeric antigen receptor is not only applicable to NK cells but can also be applied to various immune cells such as T cells, B cells, monocytes, dendritic cells, and macrophages, thereby enabling the development of personalized treatment plans based on the patient's tumor type and immune status.

[0077] According to an embodiment of the present invention, the immune cells are NK cells. Specifically, since the chimeric antigen receptor is designed primarily for NK cells, it exhibits optimal effects on NK cells.

[0078] According to embodiments of the present invention, the intracellular region of the chimeric antigen receptor is selected from the complete or partial amino acid sequences of at least two of the following proteins: 2B4, DAP10, CD3ζ, and SAP. Thus, by binding to the intracellular activation domains of at least two proteins (such as 2B4, DAP10, CD3ζ, and SAP), the chimeric antigen receptor significantly enhances CAR-mediated signal transduction, further improving the activation efficiency and anti-tumor activity of immune cells. Simultaneously, the cytokine secretion capacity and tumor cell killing ability of immune cells are also simultaneously enhanced.

[0079] According to embodiments of the present invention, the structure of the chimeric antigen receptor is as follows: the extracellular region is the extracellular domain of an NK cell activation receptor or a portion thereof, and the transmembrane region is the transmembrane region of an NK cell activation receptor or a portion thereof. The extracellular region and the transmembrane region originate from the same or different NK cell activation receptors, or the transmembrane region originates from a type I or type II transmembrane protein or a synthetically produced transmembrane protein with a hydrophobic helix. Thus, by designing the source and combination of the extracellular region and the transmembrane region, the structure of the CAR is further optimized, resulting in better performance and stability.

[0080] According to embodiments of the present invention, the extracellular region, transmembrane region, and intracellular region are directly connected or connected via linkers. This allows the connection methods between the domains to be adjusted according to different needs, thereby optimizing the performance of the chimeric antigen receptor.

[0081] According to embodiments of the present invention, the NK cell activation receptor is selected from one or more of NKG2D, DNAM-1, 2B4, NKp30, NKp46, NKp44, NKp80, NKG2C, and NKG2E. Thus, by selecting different NK cell activation receptors, the chimeric antigen receptor can be further structurally combined and its performance optimized according to different tumor types, immune cells, and drug properties.

[0082] According to an embodiment of the present invention, the NK cell activation receptor is NKG2D. Therefore, NKG2D, as one of the main activation receptors of NK cells, can recognize ligands highly expressed on various tumor cells (such as MICA / B, ULBP), exhibiting highly efficient anti-tumor activity in various tumor types. By integrating the extracellular domain of NKG2D into the chimeric antigen receptor, the immune cells and drugs are obtained, enabling them to specifically bind to tumor cells. This significantly improves the safety and efficacy of its therapeutic application while avoiding non-specific activation of normal cells.

[0083] According to an embodiment of the present invention, the segment formed by the connection of the extracellular domain and the transmembrane domain constitutes the NKG2D protein. Therefore, the chimeric antigen receptor can be obtained by connecting the extracellular domain of NKG2D to the transmembrane domain and then to the intracellular domain, thereby preparing the immune cells and drugs.

[0084] According to embodiments of the present invention, the chimeric antigen receptor is selected from any of the following structures:

[0085] (a): [SAP]-[CD3ζ-ICD]-[2B4-ICD]-[NKG2D];

[0086] (b): [SAP]-[CD3ζ-ICD]-[DAP10-ICD]-[2B4-ICD]-[NKG2D];

[0087] (c): [CD3ζ-ICD]-[t2B4-ICD]-[NKG2D];

[0088] (d): [CD3ζ-ICD]-[DAP10-ICD]-[t2B4-ICD]-[NKG2D];

[0089] (e): [SAP]-[CD3ζ-ICD]-[t2B4-ICD]-[NKG2D];

[0090] In this context, "[]" represents a structural domain, and "]-[" represents a connection between structural domains.

[0091] The connections between the structural domains are either direct connections or connections via connectors;

[0092] CD3ζ-ICD, DAP10-ICD, and 2B4-ICD represent the intracellular activation domains of the CD3ζ, DAP10, and 2B4 proteins, respectively.

[0093] t2B4-ICD is a truncated portion of the intracellular activation domain of the 2B4 protein. Thus, by providing various specific chimeric antigen receptor structural forms, diverse options are offered for different tumor types, different immune cell platforms, and different drug preparations.

[0094] According to embodiments of the present invention, the amino acid sequence of CD3ζ-ICD is shown in SEQ ID NO: 1, the amino acid sequence of 2B4-ICD is shown in SEQ ID NO: 2, the amino acid sequence of t2B4-ICD is shown in SEQ ID NO: 3, the amino acid sequence of DAP10-ICD is shown in SEQ ID NO: 4, the amino acid sequence of SAP is shown in SEQ ID NO: 5, and the amino acid sequence of NKG2D is shown in SEQ ID NO: 6.

[0095] Chimeric antigen receptor

[0096] This invention proposes a chimeric antigen receptor. According to embodiments of the invention, it includes: an extracellular region capable of binding to a tumor antigen; a transmembrane region connected to the extracellular region; and an intracellular region connected to the transmembrane region; wherein the intracellular region is selected from the complete or partial amino acid sequence of at least one of the following proteins: 2B4, DAP10, CD3ζ, and SAP; the extracellular region is derived from an NK cell activation receptor; and the tumor antigen is a tumor-specific antigen or a tumor-associated antigen. In the chimeric antigen receptor of this invention, the intracellular region integrates multiple key signal transduction elements (such as 2B4, DAP10, CD3ζ, and SAP), and the extracellular region is derived from an NK cell activation receptor (such as NKG2D), capable of specifically binding to tumor antigens. Therefore, the chimeric antigen receptor, through its innovative intracellular and extracellular segment design, significantly enhances the expression level of CAR molecules in NK cells and the NK cells' ability to recognize and attack tumor cells. Through an optimized combination of signal transduction elements (such as 2B4, DAP10, CD3ζ, and SAP), it overcomes the shortcomings of traditional NKR activation efficacy while further expanding the target selection range of CAR-NK cells. Furthermore, this CAR structural design is expected to exert highly effective therapeutic effects on various NKG2D ligand-positive tumor types, including liver cancer, breast cancer, colon cancer, and leukemia, demonstrating broad applicability. Simultaneously, its precise recognition mechanism reduces non-specific attacks on normal cells, improving its safety and efficacy in therapeutic applications. More importantly, this chimeric antigen receptor is not only applicable to NK cells but can also be applied to various immune cells such as T cells and macrophages, providing a new approach for CAR-immunotherapy with broad application prospects.

[0097] This invention was made based on the following work of the inventors:

[0098] (1) Screening of intracellular segments: In order to screen for intracellular segments that are more suitable for NK cell signal transduction, the inventors tested various combinations of signal transduction segments of various molecules such as chemokine receptors, membrane immune receptors, G protein-coupled receptors, and key proteins in signal transduction pathways. Unexpectedly, they found that the combination of DAP10, 2B4, CD3ζ and the signaling lymphocytic activation molecule-associated protein (SAP) showed a significant advantage. That is, the signal transduction segments of these elements can be linked together to form a new intracellular activation segment that is more suitable for NK cells, named NeoCAD (Neo Cytoplasmic Activation Domain).

[0099] The aforementioned NeoCAD combinations include, but are not limited to: 2B4-ICD+CD3ζ-ICD, 2B4-ICD+DAP10-ICD+CD3ζ-ICD, SAP+P2A+2B4-ICD+CD3ζ-ICD, SAP+P2A+2B4-ICD+DAP10-ICD+CD3ζ-ICD, t2B4-ICD+CD3ζ-ICD, t2B4-ICD+DAP10-ICD+CD3ζ-ICD, SAP+P2A+t2B4-ICD+CD3ζ-ICD, and combinations containing G4S links; wherein, P2A and G4S are linkers that do not affect the function of the functional amino acids themselves; t2B4-ICD is a truncated portion of the intracellular activation domain of the 2B4 protein.

[0100] (2) Identification and modification of the extracellular segment: NKG2D, as one of the most important and thoroughly studied natural killer cell receptors (NKRs) of NK cells, is believed by researchers to recognize specific ligand molecules such as MICA / B and ULBP1-6. These ligand molecules are highly expressed on tumor cells but are hardly expressed in normal tissues. However, due to its relatively short intracellular signal segment, it does not have enough structure to independently transmit activation signals. That is, when NKG2D exists alone, it is difficult to effectively convert the recognized ligand signals into NK cell activation signals. Therefore, by modifying the intracellular segment of NKG2D and combining it with additional intracellular signal modules, the activation signals can be transmitted more effectively and downstream effector functions can be exerted.

[0101] (3) Construction of CAR molecular structure: The novel intracellular activation segment NeoCAD obtained above was tandemly linked with the aforementioned NKG2D to form a series of novel CAR structural molecules that are more suitable for NK cells to be used for CAR-NK cell therapy, and named NeoN-NeoCAD.

[0102] The structure of NeoN-NeoCAD mentioned above includes, but is not limited to, the following composition (from the N-terminus to the C-terminus of the peptide chain, due to NK G2D is a type II transmembrane molecule, therefore its N-terminus is intracellular and its C-terminus is extracellular (reversing the original N-terminus to C-terminus arrangement is necessary): CD3ζ-ICD+2B4-ICD+NKG2D, CD3ζ-ICD+DAP10-ICD+2B4-ICD+NKG2D, SAP+P2A+CD3ζ-ICD+2B4-ICD+NKG2D, SAP+P2A+CD3ζ-ICD+DAP10-ICD+2B4-ICD+NKG2D, CD3ζ-ICD+t2B4-ICD+NKG2D, CD3ζ-ICD+DAP10-ICD+t2B4-ICD+NKG2D, SAP+P2A+CD3ζ-ICD+t2B4-ICD+NKG2D, etc., as well as combinations containing G4S linkages.

[0103] According to embodiments of the present invention, the intracellular region of the chimeric antigen receptor is selected from the complete or partial amino acid sequences of at least two of the following proteins: 2B4, DAP10, CD3ζ, and SAP. Thus, by combining multiple intracellular signaling proteins, the signal transduction capability of the chimeric antigen receptor is enhanced. This multi-signaling protein combination design improves the activation efficiency and persistence of immune cells, thereby more effectively killing tumor cells. Furthermore, this design allows the chimeric antigen receptor to be structurally and structurally optimized according to cell type and tumor environment.

[0104] According to embodiments of the present invention, the structure of the chimeric antigen receptor is as follows: the extracellular region is the extracellular domain of an NK cell activation receptor or a portion thereof, and the transmembrane region is the transmembrane region of an NK cell activation receptor or a portion thereof. The extracellular region and the transmembrane region originate from the same or different NK cell activation receptors, or the transmembrane region originates from a type I or type II transmembrane protein or a synthetically produced transmembrane protein with hydrophobic helices. Thus, by designing the extracellular region as the extracellular domain of an NK cell activation receptor, it can specifically bind to tumor antigens while reducing non-specific activation of normal cells, thereby achieving precise targeting of tumor cells and further improving its safety in therapeutic applications. Furthermore, the transmembrane region has diverse origins, including the NK cell activation receptor itself, other type I or type II transmembrane proteins, and even synthetically produced transmembrane proteins with hydrophobic helices. This design flexibility allows the chimeric antigen receptor to be structurally optimized according to different immune cell types and therapeutic needs.

[0105] According to embodiments of the present invention, the extracellular region, transmembrane region, and intracellular region are directly connected or connected via linkers. Thus, by directly connecting or connecting via linkers, the structural stability of the chimeric antigen receptor is optimized, signal transduction barriers caused by improper domain connection are reduced, and this flexible connection method enables the chimeric antigen receptor to be efficiently expressed in different immune cells while ensuring efficient signal transduction, further improving the applicability of the chimeric antigen receptor for different application scenarios.

[0106] It should be noted that there is no particular limitation on the selection of "linker" in this invention. All linker peptide forms known in the art that can be used to link functional amino acids without affecting the function of the functional amino acids themselves are included within the scope of protection of this invention and can be used as linkers in this invention.

[0107] According to embodiments of the present invention, the NK cell activation receptor is selected from one or more of NKG2D, DNAM-1, 2B4, NKp30, NKp46, NKp44, NKp80, NKG2C, and NKG2E. Thus, by selecting different NK cell activation receptors, it is possible to further optimize the structure and performance of chimeric antigen receptors according to different tumor types and immune cell characteristics.

[0108] According to an embodiment of the present invention, the NK cell activation receptor is NKG2D.

[0109] According to an embodiment of the present invention, the segment formed by the connection of the extracellular domain and the transmembrane domain constitutes the NKG2D protein. Therefore, the chimeric antigen receptor can be obtained by connecting the extracellular domain of NKG2D to the transmembrane domain and connecting the transmembrane domain of the NKG2D protein to a selected intracellular domain.

[0110] According to embodiments of the present invention, the chimeric antigen receptor is selected from any of the following structures:

[0111] (a): [SAP]-[CD3ζ-ICD]-[2B4-ICD]-[NKG2D];

[0112] (b): [SAP]-[CD3ζ-ICD]-[DAP10-ICD]-[2B4-ICD]-[NKG2D];

[0113] (c): [CD3ζ-ICD]-[t2B4-ICD]-[NKG2D];

[0114] (d): [CD3ζ-ICD]-[DAP10-ICD]-[t2B4-ICD]-[NKG2D];

[0115] (e): [SAP]-[CD3ζ-ICD]-[t2B4-ICD]-[NKG2D];

[0116] In this context, "[]" represents a structural domain, and "]-[" represents a connection between structural domains.

[0117] The connections between the structural domains are either direct connections or connections via connectors;

[0118] CD3ζ-ICD, DAP10-ICD, and 2B4-ICD represent the intracellular activation domains of the CD3ζ, DAP10, and 2B4 proteins.

[0119] t2B4-ICD is a truncated portion of the intracellular activation domain of the 2B4 protein. Thus, by providing multiple specific chimeric antigen receptor structural forms, diverse options are offered for different tumor types, immune cell platforms, and therapeutic needs.

[0120] According to embodiments of the present invention, the amino acid sequence of CD3ζ-ICD is shown in SEQ ID NO: 1, the amino acid sequence of 2B4-ICD is shown in SEQ ID NO: 2, the amino acid sequence of t2B4-ICD is shown in SEQ ID NO: 3, the amino acid sequence of DAP10-ICD is shown in SEQ ID NO: 4, the amino acid sequence of SAP is shown in SEQ ID NO: 5, and the amino acid sequence of NKG2D is shown in SEQ ID NO: 6.

[0121] Nucleic acid molecules

[0122] This invention proposes a nucleic acid molecule. According to embodiments of the invention, the nucleic acid molecule encodes the aforementioned chimeric antigen receptor. The nucleic acid molecule according to embodiments of the invention, which encodes the aforementioned chimeric antigen receptor, enables the aforementioned chimeric antigen receptor to be expressed efficiently and stably in immune cells. Exemplarily, the nucleic acid molecule may be a synthetically produced DNA fragment containing the complete coding sequence of the chimeric antigen receptor (e.g., the amino acid sequences of the extracellular, transmembrane, and intracellular regions), its sequence design based on specific signal transduction elements (e.g., 2B4, DAP10, CD3ζ, and SAP) and targeting domains (e.g., NKG2D) to ensure optimal function in immune cells; furthermore, the nucleic acid molecule may also contain regulatory elements such as promoters and terminators to further optimize transcription and translation efficiency.

[0123] expression carrier

[0124] This invention proposes an expression vector. According to embodiments of the invention, the expression vector comprises the aforementioned nucleic acid molecule. The expression vector according to embodiments of the invention integrates the nucleic acid molecule encoding the aforementioned chimeric antigen receptor into the expression vector, thereby enabling the chimeric antigen receptor gene to be introduced into immune cells efficiently and stably expressed in immune cells. Exemplarily, this expression vector can be an expression vector constructed based on lentivirus.

[0125] Recombinant virus

[0126] This invention proposes a recombinant virus. According to embodiments of the invention, the recombinant virus comprises the aforementioned expression vector, and the recombinant virus is capable of infecting immune cells. The recombinant virus according to embodiments of the invention can efficiently infect immune cells, thereby achieving efficient and stable expression of the aforementioned chimeric antigen receptor in immune cells, providing a method for the preparation of modified cells such as CAR-NK cells or CAR-T cells. Exemplarily, the recombinant virus of this invention can be a recombinant virus constructed based on lentivirus.

[0127] immune cells

[0128] This invention proposes an immune cell. According to embodiments of the invention, the immune cell carries the aforementioned nucleic acid molecule or the aforementioned expression vector, or is capable of expressing the aforementioned chimeric antigen receptor. The immune cell according to embodiments of the invention, by expressing the optimized aforementioned chimeric antigen receptor, significantly enhances the anti-tumor cytokine secretion capacity of immune cells (especially NK cells).

[0129] According to embodiments of the present invention, the immune cells include one or more of any of the above-mentioned immune cells derived from T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells, γδT cells, and iPSCs. Therefore, the chimeric antigen receptor design of the present invention allows for the selection of the most suitable cell platform according to different tumor types and treatment needs, further improving the flexibility and applicability of treatment.

[0130] According to an embodiment of the present invention, the immune cells are NK cells. The chimeric antigen receptor of the present invention is primarily designed for NK cells, and its expression is more effective in NK cells, thereby enhancing their anti-tumor capabilities and further improving their safety and efficacy in therapeutic applications.

[0131] Composition

[0132] This invention proposes a composition. According to embodiments of the invention, the composition contains one or more of the following: the aforementioned chimeric antigen receptor; the aforementioned nucleic acid molecule; the aforementioned expression vector; and the aforementioned immune cells. The composition according to embodiments of the invention, by providing the aforementioned chimeric antigen receptor, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned immune cells, offers a new approach for the clinical application of CAR therapy.

[0133] The amino acid sequences involved in this invention are detailed in Table 1.

[0134] Table 1. Amino acid sequences involved in this invention.

[0135]

[0136]

[0137]

[0138]

[0139]

[0140] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0141] Example 1: Screening of novel CAR structures suitable for NK cells and synthesis of a series of CAR expression plasmids

[0142] To screen for intracellular segments that better fit NK cell signaling, the inventors conducted multidimensional testing on multiple categories of signaling domains. These signaling domains encompass chemokine receptors (such as the CXCR family), transmembrane immune receptors (e.g., the CD3 family, FcεRIγ), downstream elements of G protein-coupled receptors (such as Gαi / o, Gαq), and lymphocyte adaptor proteins (such as the SLAM family). During the testing, a tandem signaling module composed of DAP10 (NKG2D co-receptor), 2B4 (SLAM-MF4 adaptor), CD3ζ (TCR signaling core), and SAP (SLAM-associated protein) was unexpectedly discovered to exhibit a significant synergistic activation effect. Specifically, this combination integrates multiple signals: co-stimulatory signals from DAP10, SLAM family receptor signaling mediated by 2B4-SAP, and potent activation signals mediated by CD3ζ. This integration demonstrates a significant advantage in activating NK cells; this discovery provides a key component scheme for the design of novel chimeric receptor intracellular segments.

[0143] Therefore, the novel intracellular activation segment NeoCAD was tandemly linked with NKG2D to form a series of novel CAR structural molecules more suitable for NK cells to use in CAR-NK cell therapy, named NeoN-NeoCAD; and an intracellular segment with the common CAR-T 4-1BB and CD3ζ tandem structure was set as a control, namely the NeoN-TCAD control experimental group, wherein the amino acid sequence of NeoN-TCAD is SEQ ID No.7; the preferred amino acid sequences of the NeoN-NeoCAD combination are SEQ ID Nos.8 to 12, and are named NeoN-NeoCAD-1 to 5 respectively.

[0144] NeoN-TCAD combination diagram and NeoN-NeoCAD-1~5 combination diagram. Figure 1 .

[0145] Subsequently, Genewiz Biotechnology Co., Ltd. was commissioned to synthesize the selected NeoN-NeoCAD-1~5 and NeoN-TCAD sequences and construct them into the pCDH vector to construct CAR molecular expression plasmids (NeoN-NeoCAD-1), (NeoN-NeoCAD-2), (NeoN-NeoCAD-3), (NeoN-NeoCAD-4), (NeoN-NeoCAD-5) and (NeoN-TCAD).

[0146] Example 2: Preparation of a series of NK cells loaded with novel CAR molecules

[0147] 1. Untreated group

[0148] NK cells in the logarithmic growth phase (purchased from Pronosai) were cultured in MEMα medium (purchased from Peiyuan Biotechnology) to obtain a concentration of 1×10⁻⁶. 6 / mL of untreated NK cells.

[0149] 2. Traditional control group

[0150] 293T cells (purchased from Pronosei) were expanded into 10cm culture dishes beforehand. Solution A was prepared by adding 15μg of expression plasmid (NeoN-TCAD), 5μg of PMD2G, 7.5μg of PSPAX2 (plasmid purchased from Qingke), and Opti MEM (purchased from Gibco) to a final volume of 375μL. Solution B was prepared by adding 345μL of Opti MEM and 30μL of Transporter 5 (purchased from Polysciences). Solution B was slowly added dropwise to solution A, mixed thoroughly, and incubated at room temperature for 20 min. The 293T cells to be transfected were then removed, and cell growth was observed under a microscope. The incubated transfection complex was then added dropwise to the cell culture dishes, gently agitated to ensure even distribution of the culture. The cells were incubated at 37℃ in a 5% CO2 incubator for 24 h, followed by medium replacement. Carefully aspirate the culture medium, then add 15 mL of preheated DMEM medium (purchased from HyClone), and return the cells to a 37°C, 5% CO2 incubator for another 48 h. Use a 10 mL syringe to aspirate the virus solution from the culture dish and filter it through a 0.45 μm filter (purchased from Millipore) into 50 mL centrifuge tubes, collecting a total of 12 mL of virus solution. Add 1 / 3 volume of 4× virus precipitation solution (purchased from Beyotime) to each tube and incubate overnight at 4°C. The next day, centrifuge at 3000×g for 30 min at 4°C. Aspirate the supernatant using a pipette pump, add 500 μL of virus resuspension (purchased from Beyotime), and gently pipette to mix the precipitate, obtaining concentrated lentivirus of the expression plasmid (NeoN-TCAD). Inoculate NK cells with the above concentrated lentivirus at MOI=20 for 48 h to obtain a concentration of 1×10⁻⁶.6 / mL of traditional control group NK cells.

[0151] 3. Experimental Group

[0152] 293T cells (purchased from Pronosei) were expanded into 10cm culture dishes beforehand. Solution A was prepared by adding 15μg of expression plasmid (NeoN-NeoCAD-1~5), 5μg of PMD2G, 7.5μg of PSPAX2 (plasmid purchased from Qingke), and Opti MEM (purchased from Gibco) to a final volume of 375μL. Solution B was prepared by adding 345μL of Opti MEM and 30μL of Transporter 5 (purchased from Polysciences). Solution B was slowly added dropwise to solution A, mixed well, and incubated at room temperature for 20 min. The 293T cells to be transfected were then removed, and cell growth was observed under a microscope. The incubated transfection complex was then added dropwise to the cell culture dishes, gently shaken to ensure even distribution of the culture on the surface, and cultured at 37℃ in a 5% CO2 incubator for 24 h before changing the medium. Carefully aspirate the culture medium, then add 15 mL of preheated DMEM medium (purchased from HyClone), and return the cells to a 37°C, 5% CO2 incubator for another 48 h. Use a 10 mL syringe to aspirate the virus solution from the culture dish and filter it through a 0.45 μm filter (purchased from Millipore) into 50 mL centrifuge tubes, collecting a total of 12 mL of virus solution. Add 1 / 3 volume of 4× virus precipitation solution (purchased from Beyotime) to each tube and incubate overnight at 4°C. The next day, centrifuge at 3000×g for 30 min at 4°C. Aspirate the supernatant using a pipette pump, add 500 μL of virus resuspension (purchased from Beyotime), and gently pipette to mix the precipitate, obtaining concentrated lentiviruses expressing CAR molecular plasmids (NeoN-NeoCAD-1~5). Inoculate NK cells with the above concentrated lentiviruses at an MOI of 20 for 48 h, obtaining concentrations of 1×10⁻⁶ for each cell type. 6 The experimental groups consisted of CAR-NK cells I (NeoN-NeoCAD-1), CAR-NK cells II (NeoN-NeoCAD-2), CAR-NK cells III (NeoN-NeoCAD-3), CAR-NK cells IV (NeoN-NeoCAD-4), and CAR-NK cells V (NeoN-NeoCAD-5) at a density of / mL.

[0153] 4. Detection of NKG2D expression level

[0154] 100 μL of untreated NK cells obtained in step 1; 100 μL of conventional control NK cells obtained in step 2; and 100 μL each of the experimental groups CAR-NK cells I, CAR-NK cells II, CAR-NK cells III, CAR-NK cells IV, and CAR-NK cells V obtained in step 3 were taken. After washing twice with PBS, the cells were resuspended in 100 μL of FACS solution (PBS containing 0.1% sodium azide and 0.4% BSA). APC-labeled anti-human NKG2D antibody (purchased from Biolegend) was added to the cell suspensions of each group according to the antibody instructions, and the cells were incubated at 4°C in the dark for 30 min. The cells were then washed twice with PBS to remove unbound antibodies, and the cells were resuspended in 200 μL of FACS solution. The fluorescence intensity of the cells in each group was analyzed by flow cytometry, and the data were processed and analyzed using FlowJo software to determine the expression level of NKG2D in each group of NK cells.

[0155] Flow cytometry analysis results of NKG2D expression in NK cells of each group are shown below. Figure 2 .

[0156] The results showed that, compared with the untreated NK cells, the NKG2D expression level of NK cells in the traditional control group and all experimental groups infected with lentivirus was significantly upregulated, indicating that a CAR-NK cell line stably expressing CAR was successfully constructed. In addition, there were significant differences in the NKG2D expression level of CAR-NK cells in different experimental groups, indicating that different CAR molecular structures affect their expression efficiency on NK cells. Among them, the NKG2D expression level of CAR-NK cells I to V was significantly higher than that of traditional control group NK cells, suggesting that novel CAR design has potential advantages in NK cell therapy applications.

[0157] Example 3: Screening of target cell lines for in vitro functional validation

[0158] To test the in vitro killing function of different CAR-NK cells, suitable target cell lines need to be screened. Ideally, target cell lines should express NKG2D ligands (such as MICA / B) at different levels to comprehensively evaluate their killing effects. Therefore, the inventors detected and analyzed the expression of MICA / B in tumor cell lines such as NCI-H716 colon cancer cells, K562 leukemia cells, MDA-MB-231 breast cancer cells, and PLC / PRF / 5 liver cancer cells. The specific steps are as follows:

[0159] Tumor cells of the following types were collected: NCI-H716 (purchased from Pronosel), K562 (purchased from Pronosel), MDA-MB-231 (purchased from Pronosel), and PLC / PRF / 5 (purchased from Pronosel). Each tumor cell was washed twice with PBS and resuspended in FACS solution for cell counting. The concentration of each tumor cell was adjusted to 1×10⁻⁶. 6 The tumor cells were divided into two groups. One group was incubated at 4°C for 30 minutes in the dark with APC-MICA / B antibody (purchased from Biolegend) according to the antibody instructions. The other group was incubated at 4°C for 30 minutes in the dark with isotype control antibody (purchased from Biolegend) according to the antibody instructions. The tumor cells were then washed twice with PBS to remove unbound antibodies. Each group of tumor cells was then resuspended in 200 μL of FACS solution. The fluorescence intensity of each group of tumor cells was analyzed using flow cytometry, and the data were processed and analyzed using FlowJo software to determine the expression level of MICA / B antigen in each group of tumor cells.

[0160] Flow cytometry analysis results of NKG2D ligand MICA / B expression in different types of tumor cells are shown below. Figure 3 .

[0161] The results showed that tumor cells K562, NCI-H716, MDA-MB-231 and PLC / PRF / 5 all expressed MICA / B antigens, and the expression abundance of MICA / B in the four tumor cell lines showed four levels from low to high, which can comprehensively represent the expression abundance of MICA / B in different tumor cells.

[0162] The above results demonstrate that tumor cell lines K562, NCI-H716, MDA-MB-231, and PLC / PRF / 5 are applicable and comprehensive potential targets for in vitro CAR-NK cell killing function testing.

[0163] Example 4: In vitro verification of the cytokine secretion function of novel CAR-NK cells

[0164] The antitumor activity of NK cells is mainly related to the cytotoxic molecules they secrete, such as granzymes and perforin, as well as cytokines such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). To quantify the in vitro functional activity of different CAR-NK cells, the inventors used flow cytometry to evaluate the cytokine secretion capacity of different CAR-NK cells in response to stimulation by tumor cell lines K562, NCI-H716, MDA-MB-231, and PLC / PRF / 5. The specific steps are as follows:

[0165] Tumor cell lines K562, NCI-H716, MDA-MB-231, and PLC / PRF / 5 were respectively 1×10⁶ cells per well. 5 The seeding density was seeded in a 96-well culture plate; the above four types of tumor cells were divided into untreated NK cells, conventional control group NK cells and experimental group CAR-NK cells according to steps 1-3 of Example 2; and according to the effector-target ratio (the ratio of the number of NK cells to the number of target cells (tumor cells)) in 1:1, the NK cells in each group in Example 2 were mixed with the four types of tumor cells respectively; on the basis of the four types of tumor cell lines, another group was set up to directly stimulate each group of NK cells with the exogenous stimulant - PMA / Ionomycin complex for 1 hour, and the amount of PMA / Ionomycin complex added was 1 μL; after each group of NK cells was co-cultured with the corresponding stimulant PMA / Ionomycin complex (or the four types of tumor cells) for 1 hour, monensin 2.5 μg was added respectively, and then each group was incubated at 37°C in the dark for 4 hours; the NK cells in each group were collected, washed with PBS, and then fixed and permeabilized, blocked non-specific binding sites and stained intracellularly respectively: (1) using 100 μL MEDIUM (1) Fix NK cells in each group (purchased from Linke Biotechnology), incubate at room temperature for 15 min, then wash each group of NK cells with PBS to remove residual fixation reagent, then resuspend each group of NK cells in 100 μL MEDIUM B permeation reagent, permeate for 15 min, then wash each group of NK cells with PBS to remove residual permeation reagent; (2) Resuspend each group of NK cells in blocking solution (100 μL), block for 15 min, then wash each group of NK cells with PBS to remove residual blocking solution; (3) Resuspend each group of NK cells in staining buffer (PBS containing 1% BSA) (purchased from Biosharp) containing flow cytometry antibodies IFN-γ (purchased from Biolegend) and TNF-α (purchased from Biolegend), respectively, and stain in the dark for 45 min. After staining, the NK cells in each group were washed with PBS, the supernatant was removed by centrifugation, and the cell pellets in each group were resuspended in PBS. The stained cells were obtained by flow cytometry, and the data were processed and analyzed using FlowJo software.

[0166] Results of in vitro cytokine secretion function tests of different NK cells are shown below. Figure 4 .

[0167] The results showed that, compared with the untreated group, the expression levels of TNF-α and IFN-γ in NK cells of both the traditional control group and the experimental group were significantly increased after stimulation by four target cells (K562, NCI-H716, MDA-MB-231 and PLC / PRF / 5) or by the PMA / Ionomycin complex. Among them, the expression levels of TNF-α and IFN-γ in CAR-NK cells of most experimental groups were significantly higher than those in the traditional control group, indicating that the novel CAR molecule design of the present invention further enhances the functional activity of NK cells and exhibits superior performance.

[0168] Example 5: Functional validation of the novel CAR molecular structure in T cells (Jurkat cells)

[0169] To evaluate whether the novel CAR molecular structure of this invention can enhance the function of CAR-T cells in vivo and in vitro, the inventors selected NeoN-NeoCAD-4 as a representative and tested its performance in the T cell line Jurkat. The specific steps are as follows:

[0170] 1. Preparation and activation of Jurkat cells

[0171] Jurkat cells in logarithmic growth phase (purchased from Pronosei) were harvested and the cell density was adjusted to 1 x 10⁻⁶. 6 Jurkat cells / mL; Take a 6-well plate, add 4 mL of Jurkat cells at the above cell density to each well, and then add 40 μL of TransAct reagent (purchased from Miltenyi) and interleukin-2 (IL-2) (purchased from Tetracycline Biotech) to each well to activate Jurkat cells.

[0172] 2. Untreated group

[0173] The activated Jurkat cells obtained in step 1 were cultured in 1640 medium (purchased from HyClone) to obtain a concentration of 1×10⁻⁶. 6 / mL of untreated Jurkat cells.

[0174] 3. Traditional control group

[0175] Take the activated Jurkat cells obtained in step 1, add concentrated lentivirus containing the expression plasmid (NeoN-TCAD) prepared in Example 2 to the Jurkat cells at an MOI of 20, and add the infection-promoting reagent polybrene (1:1000). Infect for 48 hours, then add 2 μg / mL puromycin and continue for 2 weeks. Select Jurkat cells successfully transfected with the aforementioned target vector, and finally obtain a concentration of 1×10⁻⁶. 6 / mL of traditional control group Jurkat cells.

[0176] 4. Experimental Group

[0177] Take the activated Jurkat cells obtained in step 1, add concentrated lentivirus containing the CAR expression plasmid (NeoN-NeoCAD-4) prepared in Example 2 to the Jurkat cells at an MOI of 20, and add the infection-promoting reagent polybrene (1:1000). Infect for 48 hours, then add 2 μg / mL puromycin and continue for 2 weeks. Select Jurkat cells successfully transfected with the aforementioned target vector, and finally obtain cells with a concentration of 1×10⁻⁶. 6 Jurkat cells in the experimental group ( / mL).

[0178] 5. In vitro cytokine secretion capacity assay

[0179] Tumor cell lines K562, NCI-H716, MDA-MB-231, and PLC / PRF / 5 were respectively 1×10⁶ cells per well. 5The cells were seeded at a density of 100 cells / wells in 96-well culture plates. The four types of tumor cells were divided into three groups according to steps 2-4 of Example 5: untreated Jurkat cells, conventional control Jurkat cells, and experimental Jurkat cells. The Jurkat cells obtained in steps 2-4 of Example 5 were then co-cultured with the four types of tumor cells at a 1:1 effector-to-target ratio (the ratio of Jurkat cells to target cells (tumor cells)). In addition to the four tumor cell lines, a separate group was set up where each group of NK cells was directly stimulated for 1 hour with an exogenous stimulant—the PMA / Ionomycin complex—at an amount of 1 μL. After co-culturing each group of Jurkat cells with the corresponding stimulant PMA / Ionomycin complex (or 4 types of tumor cells) for 1 hour, monensin 2.5 μg was added to each group, and then each group was incubated at 37°C in the dark for 4 hours. The Jurkat cells of each group were collected, washed with PBS, and then fixed and permeabilized, blocked non-specific binding sites and intracellular staining were performed respectively: (1) The Jurkat cells of each group were fixed with 4% paraformaldehyde for 15 min, and then washed with PBS to remove residual fixation reagent. Then the NK cells of each group were resuspended in 100 μL of permeabilization reagent 1% Triton. (1) Permeabilize the cells in X-100 for 15 min, then wash the Jurkat cells in each group with PBS to remove residual permeabilizing reagent; (2) Resuspend the Jurkat cells in each group in blocking buffer CD16 / 32 antibody (Biolegend), block for 15 min, then wash the Jurkat cells in each group with PBS to remove residual blocking buffer; (3) Resuspend the Jurkat cells in each group in staining buffer (PBS contains 1% BSA) containing flow cytometry antibodies IFN-γ (purchased from Biolegend) and TNF-α (purchased from Biolegend), respectively, and stain in the dark for 30 min. After staining, wash the Jurkat cells in each group with PBS, centrifuge to remove supernatant, and finally resuspend the Jurkat cell pellet in PBS. Obtain stained cells using flow cytometry and process and analyze the data using FlowJo software.

[0180] Results of in vitro cytokine secretion function tests of different T cell (Jurkat) cells are shown in [the table below]. Figure 5 .

[0181] The results showed that, compared with the untreated group, the expression levels of TNF-α and IFN-γ in Jurkat cells in both the traditional control group and the experimental group were significantly increased after stimulation by four target cells (K562, NCI-H716, MDA-MB-231 and PLC / PRF / 5) or by the PMA / Ionomycin complex. In particular, the expression levels of TNF-α and IFN-γ in the experimental group of Jurkat cells were significantly higher than those in the traditional control group, indicating that the novel CAR molecule design of this invention further enhances the functional activity of Jurkat cells and exhibits superior performance.

[0182] This embodiment exemplifies the in vitro secretion of cytokines by Jurkat cells in the NeoN-NeoCAD-4 experimental group. Cells in other experimental groups (NeoN-NeoCAD-1, NeoN-NeoCAD-2, NeoN-NeoCAD-3, and NeoN-NeoCAD-5) also showed the same enhanced efficacy.

[0183] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0184] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of an immune cell in the manufacture of a medicament, characterized in that, The medicine is capable of improving the anti-tumor cytokine secretion ability of the immune cells; The immune cells are modified with a chimeric antigen receptor, which comprises an extracellular region, a transmembrane region and an intracellular region; The extracellular region is capable of binding to a tumor antigen; The transmembrane region is connected to the extracellular region; The intracellular region is connected to the transmembrane region; The intracellular region is selected from the entire amino acid sequence or partial amino acid sequence of at least one of the following proteins: 2B4, DAP10, CD3ζ and SAP; The extracellular region is derived from an NK cell activating receptor; The tumor antigen is a tumor specific antigen or a tumor associated antigen.

2. Use according to claim 1, characterized in that, The anti-tumor cytokine includes one or more of interferon gamma, tumor necrosis factor alpha and interleukin.

3. Use according to claim 1, characterized in that, The immune cells include one or more of T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells, γδ T cells and iPSC-derived immune cells of any of the foregoing; Optionally, the immune cells are NK cells.

4. Use according to claim 1, characterized in that, The intracellular region of the chimeric antigen receptor is selected from the entire amino acid sequence or partial amino acid sequence of at least two of the following proteins: 2B4, DAP10, CD3ζ and SAP.

5. Use according to claim 1, characterized in that, The structure of the chimeric antigen receptor is as follows: The extracellular region is an extracellular domain of an NK cell activating receptor or a part thereof, the transmembrane region is a transmembrane region of an NK cell activating receptor or a part thereof, the NK cell activating receptors from which the extracellular region and the transmembrane region are derived are the same or different, or the transmembrane region is derived from a type I or type II transmembrane protein or an artificially synthesized transmembrane protein with a hydrophobic helix.

6. Use according to claim 1, characterized in that, The extracellular region, the transmembrane region and the intracellular region are directly connected or connected through a linker; Optionally, the NK cell activating receptor is selected from one or more of NKG2D, DNAM-1, 2B4, NKp30, NKp46, NKp44, NKp80, NKG2C and NKG2E; Optionally, the NK cell activating receptor is NKG2D; Optionally, the segment after the extracellular region is connected to the transmembrane region constitutes an NKG2D protein.

7. Use according to claim 1, characterized in that, The chimeric antigen receptor is selected from any of the following structures: (a): [SAP]-[CD3ζ-ICD]-[2B4-ICD]-[NKG2D]; (b): [SAP]-[CD3ζ-ICD]-[DAP10-ICD]-[2B4-ICD]-[NKG2D]; (c): [CD3ζ-ICD]-[t2B4-ICD]-[NKG2D]; (d): [CD3ζ-ICD]-[DAP10-ICD]-[t2B4-ICD]-[NKG2D]; (e): [SAP]-[CD3ζ-ICD]-[t2B4-ICD]-[NKG2D]; Wherein, "[]" represents a domain, and "]-[" represents the connection between domains; The connection between the domains is a direct connection or a connection through a linker; CD3ζ-ICD, DAP10-ICD, 2B4-ICD represent the intracellular activation domain of CD3ζ, DAP10, 2B4 protein; t2B4-ICD is a part of the intracellular activation domain of the 2B4 protein after truncation processing.

8. Use according to claim 1, characterized in that, The amino acid sequence of CD3ζ-ICD is shown as SEQ ID NO: 1, the amino acid sequence of 2B4-ICD is shown as SEQ ID NO: 2, the amino acid sequence of t2B4-ICD is shown as SEQ ID NO: 3, the amino acid sequence of DAP10-ICD is shown as SEQ ID NO: 4, the amino acid sequence of SAP is shown as SEQ ID NO: 5, and the amino acid sequence of NKG2D is shown as SEQ ID NO:

6.

9. A chimeric antigen receptor, characterized in that, It comprises: an extracellular region capable of binding to a tumor antigen; a transmembrane region connected to the extracellular region; an intracellular region connected to the transmembrane region; wherein the intracellular region is selected from the entire amino acid sequence or part of the amino acid sequence of at least one of the following proteins: 2B4, DAP10, CD3ζ and SAP; The extracellular region is derived from an NK cell activating receptor. The tumor antigen is a tumor specific antigen or a tumor associated antigen.

10. The chimeric antigen receptor of claim 9, wherein, The intracellular region of the chimeric antigen receptor is selected from the entire amino acid sequence or part of the amino acid sequence of at least two of the following proteins: 2B4, DAP10, CD3ζ and SAP.

11. The chimeric antigen receptor of claim 9, wherein, The structure of the chimeric antigen receptor is as follows: The extracellular region is the extracellular domain or a part of the extracellular domain of an NK cell activating receptor, and the transmembrane region is the transmembrane region or a part of the transmembrane region of an NK cell activating receptor. The NK cell activating receptors from which the extracellular region and the transmembrane region are derived are the same or different, or the transmembrane region is derived from a type I or type II transmembrane protein or an artificially synthesized transmembrane protein with a hydrophobic helix.

12. The chimeric antigen receptor of claim 9, wherein, The extracellular region, transmembrane region and intracellular region are directly connected or connected through a linker; Optionally, the NK cell activating receptor is selected from one or more of NKG2D, DNAM-1, 2B4, NKp30, NKp46, NKp44, NKp80, NKG2C and NKG2E; Optionally, the NK cell activating receptor is NKG2D; Optionally, the segment after the connection of the extracellular region and the transmembrane region constitutes an NKG2D protein.

13. The chimeric antigen receptor of claim 9, wherein, The chimeric antigen receptor is selected from any of the following structures: (a): [SAP]-[CD3ζ-ICD]-[2B4-ICD]-[NKG2D]; (b): [SAP]-[CD3ζ-ICD]-[DAP10-ICD]-[2B4-ICD]-[NKG2D]; (c): [CD3ζ-ICD]-[t2B4-ICD]-[NKG2D]; (d): [CD3ζ-ICD]-[DAP10-ICD]-[t2B4-ICD]-[NKG2D]; (e): [SAP]-[CD3ζ-ICD]-[t2B4-ICD]-[NKG2D]; wherein, "[]" represents a domain, and "] - [" represents a connection between domains; the connection between the domains is a direct connection or a connection through a linker; CD3 zeta-ICD, DAP10-ICD, 2B4-ICD represents the intracellular activation domain of CD3 zeta, DAP10, 2B4 protein; t2B4-ICD is a part of the intracellular activation domain of the 2B4 protein after truncation processing.

14. The chimeric antigen receptor of claim 9, wherein, The amino acid sequence of the CD3 zeta-ICD is shown as SEQ ID NO: 1, the amino acid sequence of the 2B4-ICD is shown as SEQ ID NO: 2, the amino acid sequence of the t2B4-ICD is shown as SEQ ID NO: 3, the amino acid sequence of the DAP10-ICD is shown as SEQ ID NO: 4, the amino acid sequence of the SAP is shown as SEQ ID NO: 5, and the amino acid sequence of the NKG2D is shown as SEQ ID NO:

6.

15. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the chimeric antigen receptor of any one of claims 9-14.

16. An expression vector comprising the nucleic acid of claim 15. The expression vector comprises the nucleic acid molecule of claim 15.

17. A recombinant virus, wherein, The recombinant virus comprises the expression vector of claim 16, and the recombinant virus is capable of infecting immune cells.

18. An immune cell, comprising, The immune cells carry the nucleic acid molecule of claim 15 or the expression vector of claim 8 or are capable of expressing the chimeric antigen receptor of any one of claims 9-14.

19. The immune cell of claim 18, wherein, The immune cells include one or more of T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells, gamma delta T cells, and iPSC-derived immune cells. Optionally, the immune cells are NK cells.

20. A composition characterized in that, The composition contains one or more of the following: the chimeric antigen receptor of any one of claims 9-14; the nucleic acid molecule of claim 15; the expression vector of claim 16; the immune cells of claim 18 or 19.

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