CAR (chimeric antigen receptor) structure containing green fluorescent protein as well as preparation method and application of CAR structure

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a CAR structure containing green fluorescent protein as well as a preparation method and application of the CAR structure. The novel chimeric antibody receptor structure provided by the invention comprises a gene segment of green fluorescent protein, and the gene segment of the green fluorescent protein is connected with a chimeric antigen receptor segment through a P2A sequence. According to the invention, a green fluorescent protein fragment is connected to a chimeric antigen receptor fragment, so that the in-vitro anti-tumor effect of CAR-T and CAR-NK can be obviously improved.

Description

[0001] Priority application This application claims priority to the Chinese invention patent application [CN2023115954384] "A CAR-T containing green fluorescent protein and preparation method and application" filed on November 24, 2023, which is incorporated by reference in its entirety. Technical Field

[0002] The present invention belongs to the field of biomedicine technology, and specifically relates to a CAR structure containing green fluorescent protein, a preparation method and an application thereof. Background Art

[0003] For tumors, traditional surgical removal, chemotherapy, and radiotherapy can no longer meet the purpose of effective monitoring and treatment, and effective alternative therapies are urgently needed to overcome this problem. In recent years, with the emergence of immunotherapy, revolutionary progress has taken place in the field of tumor treatment, such as immune checkpoint inhibitors, bispecific antibodies, and CAR-T cell therapy have achieved remarkable results.

[0004] The first generation of CARs only have the signal transduction domains of CD3ξ (containing 3 ITAMs) and FcRγ (containing 2 ITAMs) in the cell, which can only provide the first signal for T cell activation. Early clinical trials of the first generation of CARs showed that they could not effectively maintain the proliferation and activation of T cells, and did not achieve satisfactory results, indicating that the first generation of CAR-T cells may lack sufficient activation signals to maintain T cell proliferation and effective anti-tumor effects. Researchers further connected the signal domains of co-stimulatory molecules (such as CD28, 4-1BB (CD137), ICOS or OX40 (CD134)) in series on the basis of the first generation of CAR-T intracellular signal sequences to provide additional stimulation signals for T cell activation, forming the second generation of CAR-T. The second generation of CAR T cells showed enhanced in vivo expansion and persistence, and its effect has been confirmed in clinical trials.

[0005] Since then, CAR-T related technologies have been continuously innovated, but of the 939 immune cell therapy trials launched since 1993, only about half are for solid tumors. However, 90% of the global cancer incidence is solid tumors. Unfortunately, compared with the good efficacy shown in blood tumors, most reported effects of using CAR-T cells to treat solid tumors have always been difficult to effectively break through at certain key stages, so the overall efficacy of treating solid tumors is not ideal. Therefore, there is an urgent need to develop new CAR structures with effective killing capabilities to make breakthroughs in CAR-T therapy in solid tumors.

[0006] Enhanced green fluorescent protein (EGFP), also known as green fluorescent protein, is a protein produced by its gene that emits green fluorescence when excited by light in the blue wavelength range. Due to its characteristics such as spontaneous fluorescence, EGFP has been widely used in fields such as molecular biology and cell biology, but its application is more focused on using EGFP as a tool molecule. For example, as a reporter molecule, EGFP plays an important role in detecting protein expression, protein and cell fluorescence tracing, and studying protein interactions and conformational changes. So far, there have been no reports on the application of EGFP as a functional immune regulator. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide a CAR structure containing green fluorescent protein, a preparation method and an application. In the present invention, the green fluorescent protein is used as an immunomodulatory factor, which can partially alleviate or approach the problems existing in the prior art. The present invention specifically adopts the following technical solutions.

[0008] A novel chimeric antigen receptor structure, the novel chimeric antibody receptor structure comprises a gene fragment of green fluorescent protein, the gene fragment of green fluorescent protein is connected to a chimeric antigen receptor fragment through a P2A sequence; the nucleotide sequence of the green fluorescent protein is shown in SEQ ID NO.1; the chimeric antigen receptor fragment comprises a CD8 signal peptide, an antigen recognition region (SCFV), a hinge region, a human CD8 transmembrane region, a human 4-1BB co-stimulatory signal region and a human CD3ζ signal domain.

[0009] It is understandable that the above-mentioned antigen recognition region can target CD19, BCMA, HER2, Claudin18.2, Mesothelin, GPC3, GD2, etc.

[0010] Preferably, the nucleotide sequence of the chimeric antigen receptor fragment is shown in SEQ ID NO.3.

[0011] Furthermore, the novel chimeric antigen receptor is optionally connected to the EF1-α promoter sequence to initiate gene transcription and expression. A CAR-T cell expressing the novel chimeric antigen receptor structure.

[0012] A CAR-NK cell expressing the novel chimeric antigen receptor structure.

[0013] NK cells are important members of the innate immune system. They are mainly found in the blood and lymphoid organs. They do not require pre-sensitization and are not restricted by the major histocompatibility complex (MHC). They can quickly and directly kill target cells and have a broad spectrum of anti-tumor effects. Mature NK cells retain a large number of cytotoxic granules containing perforin and granzymes, as well as effector mRNA that can be translated after stimulation. NK cells kill target cells in a variety of ways. Once the balance between inhibitory and activating signals in NK cells is biased towards activation, NK cells can form synapses with target cells, thereby releasing effector granules to lyse target cells and produce effector cytokines such as IFN-γ. The expression of chimeric antigen receptors (CARs) on the surface of NK cells can significantly improve the anti-cancer effect of immune cells. Compared with CAR-T cells, CAR-NK cells have some significant advantages, including: (1) less cytokine release syndrome and neurotoxicity. (2) CAR-NK cells can kill cancer cells through both CAR-dependent and CAR-independent pathways. In addition, NK cells can kill tumor cells through CD16-mediated ADCC. Therefore, NK cells have a broad spectrum of highly effective tumor-killing activity. (3) Allogeneic re-infusion therapy can be achieved.

[0014] A drug for treating solid tumors, comprising the above-mentioned CAR-T cells.

[0015] As a preference, other pharmaceutically acceptable carriers and / or adjuvants may also be included.

[0016] A drug for treating solid tumors, comprising the above-mentioned CAR-NK cells.

[0017] As a preference, other pharmaceutically acceptable carriers and / or adjuvants may also be included.

[0018] Furthermore, the solid tumor includes ovarian cancer, lung cancer or breast cancer.

[0019] A green fluorescent protein reagent is used in the preparation of a chimeric antigen receptor immune activator, wherein the green fluorescent protein reagent comprises a nucleotide as shown in SEQ ID NO.1.

[0020] Furthermore, the chimeric antigen receptor immune activator also contains a lentiviral expression vector.

[0021] The method for preparing the novel chimeric antigen receptor structure comprises the following steps: S01: constructing an expression vector for expressing a novel chimeric antigen receptor structure, comprising a chimeric antigen receptor fragment and a green fluorescent protein gene fragment, wherein the chimeric antigen receptor fragment and the green fluorescent protein gene fragment are connected by a P2A peptide; S02: The expression vector constructed in S01 was used as the core plasmid, and psPAX2 and pMD2.G were used as auxiliary plasmids for co-transfection.

[0022] In some preferred embodiments, the expression vector is a lentiviral expression vector, but is not limited thereto.

[0023] Beneficial effects: The present invention provides a method for constructing a novel CAR structure. The present invention connects a fluorescent protein EGFP to CAR for the first time. EGFP is a GFP mutant protein, which was first derived from the Victoria jellyfish and only has the function of luminescence, and has no other biological activity. The present invention connects a fragment of EGFP to CAR for the first time, which can enhance the in vitro anti-tumor effect of CAR-T / CAR-NK. The CAR structure can be used to prepare novel cell therapy products such as CAR-T and CAR-NK.

[0024] The present invention prepares a novel CAR-NK cell, and the NK cell has a broad-spectrum and highly efficient tumor-killing activity, so the CAR-NK cell can be effective against a variety of solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0026] Figure 1 A schematic diagram of the structure of the novel CAR provided by the present invention; Figure 2 To express the CAR positive rate of the novel CAR-T of the present invention; Figure 3 These are the killing effect diagrams of the control group CAR-T and the novel CAR-T of the present invention co-cultured with tumor cells respectively; Figure 4 The killing curves of the control group CAR-T and the novel CAR-T of the present invention co-cultured with tumor cells respectively; Figure 5 The killing curves of the control group CAR-NK and the novel CAR-NK of the present invention co-cultured with tumor cells respectively; Figure 6 The supernatant of the control group CAR-T and the novel CAR-T of the present invention after co-culture with tumor cells was detected; Figure 7 The supernatant of the control group CAR-NK and the novel CAR-NK of the present invention after co-culture with tumor cells was detected. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0028] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0029] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0030] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0031] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0032] Example 1 This example provides an example of a novel chimeric antibody receptor structure.

[0033] A novel chimeric antibody receptor structure, wherein the novel chimeric antibody receptor is connected to green fluorescent protein (EGFP) via a P2A peptide, and the novel chimeric antibody receptor is optionally connected to an EF1α promoter sequence. The novel chimeric antibody receptor structure is as follows Figure 1 shown.

[0034] Green fluorescent protein is a protein isolated from the Victoria jellyfish. It is a protein composed of about 238 amino acids. It can be excited by light from blue to ultraviolet light to emit green fluorescence. This protein is mostly used for cell labeling. The present invention is the first to use a fragment of it to enhance the function of CAR.

[0035] The CAR fragment in the novel chimeric antibody receptor structure may optionally include a CD8 signal peptide, an antigen recognition region (SCFV), a hinge region (hinge), a human CD8 transmembrane region (TM), a human 4-1BB co-stimulatory signal region, and a human CD3ζ signal domain.

[0036] It will be appreciated that the CAR fragment in the novel chimeric antibody receptor structure is merely an example and not a limitation.

[0037] Example 2 This example provides an example of a method for constructing a novel chimeric antibody receptor structure (referred to as the novel CAR structure).

[0038] 1.1 Construction of novel chimeric antibody receptor structure and viral packaging.

[0039] A molecular cloning method was used to construct a lentiviral expression vector pWPXLD-CAR-P2A-X1 expressing a novel CAR structure, in which the CAR fragment contained a CD8 signal peptide, SCFV, a hinge region, a human CD8 transmembrane region (TM), a human 4-1BB co-stimulatory signal region, and a human CD3ζ signal domain, and was connected to the X1 fragment via P2A, where X1 represents green fluorescent protein.

[0040] The structural sequences involved in this embodiment are shown in Table 1.

[0041] Table 1 1.2 Packaging of lentivirus containing novel CAR structure.

[0042] Take 293T cells with good growth status, digest the cells with trypsin, and pass them at a density of 4*10^6 cells / dish. When the cell confluence reaches 80%, use the CAR vector as the core plasmid, psPAX2 and pMD2.G as auxiliary plasmids, and use calcium phosphate transfection technology for transfection. 8 hours after transfection, discard the medium containing calcium phosphate precipitation. After 48 and 72 hours of transfection, collect the cell culture supernatant as the virus stock solution, centrifuge at 2000rpm for 10 minutes, remove the cell precipitate, and use a 0.22μm disposable syringe filter (PES membrane) to remove cell debris.

[0043] Example 3 Detection of the positive rate of the novel CAR structure synthesized in Example 2.

[0044] The lentivirus in Example 2 was used to transduce T cells to obtain novel CAR-T cells. The target protein expressing the His tag was used as the primary antibody, and the His antibody coupled to the fluorescent APC was used as the secondary antibody to detect the CAR positive rate. A control group was set up, and the control group was a CAR without green fluorescent protein, and its structure was the same as the CAR shown in Example 2. The results are shown in FIG. Figure 2 shown.

[0045] The results showed that the positive expression rate of CAR could be increased after connection with green fluorescent protein.

[0046] Example 4 The tumor killing ability of the new CAR structure synthesized in Example 2 was verified.

[0047] CAR target-positive SK-OV3 cells (human ovarian cancer cells) were used as target cells, and CAR-T cells were used as effector cells. The two types of cells were co-cultured at an effector-target ratio of 5:1, and the killing effect of CAR-T cells was monitored.

[0048] Under the microscope, it can be seen that CAR-T cells containing GFP short peptide can better gather tumor cells together for killing ( Figure 3 ).

[0049] In addition, the killing effect of the killing curve for 40 hours was analyzed, and the results showed that the killing effect of CAR-peptide was significantly better than that of the control group CAR ( Figure 4 ); From about 10 hours onwards, it can be seen that the killing ability of CAR-peptide on tumor cells is significantly higher than that of the control group CAR, and its killing efficiency is higher than that of the control group and the killing time is earlier than that of the control group.

[0050] The lentivirus in Example 2 was further used to transduce NK cells to obtain novel CAR-NK cells. SK-OV3 cells with positive CAR targets were used as target cells, and CAR-NK cells were used as effector cells. The two cells were co-cultured at an effector-target ratio of 5:1 to monitor the killing of CAR-T cells.

[0051] The killing effect of the killing curve for 30 hours was analyzed, and the results showed that the killing ability of CAR-peptide on tumor cells was higher than that of the control group CAR ( Figure 5 ).

[0052] Example 4 The CAR-T and CAR-NK of Example 3 were further verified.

[0053] The supernatants of CAR-T cells and CAR-NK cells were collected after co-culture with tumor cells for 24 hours, and IFN-γ, IL-2, and TNF-α in the supernatants were analyzed. After co-culture of CAR-T cells with target cells, IFN-γ and IL-2 in the supernatants increased significantly ( Figure 6 ), IFN-γ and TNF-α in the supernatant increased significantly after CAR-NK was co-cultured with target cells ( Figure 7 ), the specific results are shown in the following table.

[0054] Table 2 CAR-T cell detection results The results showed that green fluorescent protein, as an immunomodulatory factor, significantly increased the expression of IFN-γ and IL-2 in CAR-T cells compared with the control group.

[0055] Table 3 CAR-NK cell detection results The results showed that green fluorescent protein, as an immunomodulatory factor, significantly increased the expression of IFN-γ and TNF-α in CAR-NK cells compared with the control group. The CAR-NK cells in the control group hardly expressed TNF-α.

[0056] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0057] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A novel chimeric antigen receptor structure, characterized in that: The novel chimeric antibody receptor structure comprises a gene fragment of green fluorescent protein, which is connected to a chimeric antigen receptor fragment through a P2A sequence; the nucleotide sequence of the green fluorescent protein is shown in SEQ ID NO.1; the chimeric antigen receptor fragment comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB co-stimulatory signal region and a human CD3ζ signal domain.

2. The novel chimeric antigen receptor structure according to claim 1, characterized in that: The nucleotide sequence of the chimeric antigen receptor fragment is shown in SEQ ID NO.

3.

3. The novel chimeric antigen receptor structure according to claim 1, characterized in that: The novel chimeric antigen receptor is also linked to the EF1-α promoter sequence.

4. A CAR-T cell expressing the novel chimeric antigen receptor structure according to any one of claims 1 to 3.

5. A CAR-NK cell expressing the novel chimeric antigen receptor structure according to any one of claims 1 to 3.

6. A drug for treating solid tumors, characterized in that: Comprising the CAR-T cell of claim 4.

7. A drug for treating solid tumors, characterized in that: Comprising the CAR-NK cell of claim 5.

8. Use of a green fluorescent protein reagent in the preparation of a chimeric antigen receptor immune activator, characterized in that: The green fluorescent protein reagent comprises the nucleotides shown in SEQ ID NO.

1.

9. The use according to claim 8, characterized in that The chimeric antigen receptor immune activator also contains a lentiviral expression vector.

10. The method for preparing the novel chimeric antigen receptor structure according to claim 1, characterized in that: The following steps are involved: S01: constructing an expression vector for expressing a novel chimeric antigen receptor structure, comprising a chimeric antigen receptor fragment and a green fluorescent protein gene fragment, wherein the chimeric antigen receptor fragment and the green fluorescent protein gene fragment are connected by a P2A peptide; S02: The expression vector constructed in S01 was used as the core plasmid, and psPAX2 and pMD2.G were used as auxiliary plasmids for co-transfection.

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