A CAR structure containing green fluorescent protein and its preparation method and application

Abstract

The present invention belongs to the field of biomedicine and specifically relates to a CAR structure containing green fluorescent protein, as well as its preparation method and application. The novel chimeric antibody receptor structure provided by the present invention comprises a gene fragment for green fluorescent protein, which is linked to a chimeric antigen receptor fragment via a P2A sequence. The present invention, by linking a green fluorescent protein fragment to a chimeric antigen receptor fragment, can significantly enhance the in vitro anti-tumor effects of CAR-T and CAR-NK.

Description

[0001] Priority application

[0002] This application claims priority to the Chinese invention patent application [CN2023115954384] "A CAR-T containing green fluorescent protein and its preparation method and application" filed on November 24, 2023, which is incorporated by reference in its entirety. Technical Field

[0003] 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

[0004] Traditional methods of surgical removal, chemotherapy, and radiotherapy are no longer sufficient for effective monitoring and treatment of tumors, and effective alternative therapies are urgently needed to overcome this challenge. In recent years, with the emergence of immunotherapy, revolutionary advances have taken place in the field of tumor treatment, with immune checkpoint inhibitors, bispecific antibodies, and CAR-T cell therapies all achieving remarkable results.

[0005] First-generation CARs contain only the intracellular signaling domains of CD3ζ (containing three ITAMs) and FcRγ (containing two ITAMs), providing only the initial signal for T cell activation. Early clinical trials of first-generation CARs demonstrated an inability to effectively maintain T cell proliferation and activation, resulting in unsatisfactory results. This suggests that first-generation CAR-T cells may lack sufficient activation signals to maintain T cell proliferation and effective anti-tumor effects. Researchers have further cascaded the signaling domains of costimulatory molecules (such as CD28, 4-1BB (CD137), ICOS, or OX40 (CD134)) onto the intracellular signaling sequence of first-generation CAR-T cells, providing additional stimulatory signals for T cell activation, resulting in the development of second-generation CAR-T cells. Second-generation CAR T cells have demonstrated enhanced in vivo expansion and persistence, and their efficacy has been confirmed in clinical trials.

[0006] CAR-T technology has been continuously innovated since then, but of the 939 immune cell therapy trials initiated since 1993, only approximately half have targeted solid tumors. However, solid tumors account for 90% of global cancer incidence. Unfortunately, compared to the promising results demonstrated in hematologic malignancies, most reported CAR-T cell therapies for solid tumors have struggled to achieve significant breakthroughs at certain critical stages, resulting in less than ideal overall efficacy in treating solid tumors. Therefore, the development of new CAR structures with effective killing capabilities is urgently needed to achieve breakthroughs in CAR-T therapy for solid tumors.

[0007] Enhanced green fluorescent protein (EGFP), also known as green fluorescent protein, produces a protein that emits green fluorescence when stimulated by light in the blue wavelength range. Due to its properties, such as autofluorescence, EGFP has been widely used in fields such as molecular biology and cell biology, but its applications are primarily focused on its use as a tool molecule. For example, as a reporter molecule, EGFP plays an important role in monitoring protein expression, fluorescent tracing of proteins and cells, and studying protein interactions and conformational changes. However, there are currently no reports on the use of EGFP as a functional immunomodulator. Summary of the Invention

[0008] 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, 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.

[0009] A novel chimeric antigen receptor structure, the novel chimeric antibody receptor structure comprises a gene fragment of green fluorescent protein, wherein the gene fragment of green fluorescent protein is connected to the chimeric antigen receptor fragment via 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.

[0010] It is understandable that the above-mentioned antigen recognition region can target CD19, BCMA, HER2, Claudin18.2, Mesothelin, GPC3, GD2, etc.

[0011] Preferably, the nucleotide sequence of the chimeric antigen receptor fragment is shown in SEQ ID NO.3.

[0012] Furthermore, the novel chimeric antigen receptor is optionally connected to the EF1-α promoter sequence to initiate gene transcription and expression.

[0013] A CAR-T cell expressing the novel chimeric antigen receptor structure.

[0014] A CAR-NK cell expressing the novel chimeric antigen receptor structure.

[0015] 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 signals and activation 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. By expressing chimeric antigen receptors (CARs) on the surface of NK cells, the anti-cancer effect of immune cells can be significantly improved. 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 tumoricidal activity. (3) Allogeneic transfusion therapy can be achieved.

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

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

[0018] A drug for treating solid tumors, comprising the above-mentioned CAR-NK cells.

[0019] As a preference, other pharmaceutically acceptable carriers and / or adjuvants may also be included.

[0020] Furthermore, the solid tumor includes ovarian cancer, lung cancer or breast cancer.

[0021] A green fluorescent protein reagent is used in the preparation of a chimeric antigen receptor immune activator. The green fluorescent protein reagent comprises a nucleotide as shown in SEQ ID NO.1.

[0022] Furthermore, the chimeric antigen receptor immune activator also contains a lentiviral expression vector.

[0023] The method for preparing the novel chimeric antigen receptor structure comprises the following steps:

[0024] 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;

[0025] 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.

[0026] In some preferred embodiments, the expression vector is a lentiviral expression vector, but is not limited thereto.

[0027] Beneficial effects:

[0028] This invention provides a method for constructing a novel CAR structure. For the first time, the fluorescent protein EGFP is linked to the CAR. EGFP is a GFP mutant originally derived from the jellyfish Aequorea victoria. It only emits light and has no other biological activity. This method, for the first time, links an EGFP fragment to the CAR, potentially enhancing the in vitro anti-tumor efficacy of CAR-T / CAR-NK. This CAR structure can be used to prepare novel cell therapy products such as CAR-T and CAR-NK.

[0029] The present invention prepares a new type of CAR-NK cell. The NK cell has a broad spectrum and efficient tumor killing activity, so the CAR-NK cell can be effective against a variety of solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the embodiments or the description of the prior art. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the various elements or parts are 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 inventive work.

[0031] Figure 1 This is a schematic diagram of the structure of the novel CAR provided by the present invention;

[0032] Figure 2 The CAR positive rate of the novel CAR-T cell expressing the present invention;

[0033] Figure 3 Figure 2 is the killing effect of the control group CAR-T and the novel CAR-T of the present invention co-cultured with tumor cells;

[0034] Figure 4 Figure 2 is a killing curve of the control group CAR-T and the novel CAR-T of the present invention co-cultured with tumor cells;

[0035] Figure 5 Figure 3 is a killing curve of the control group CAR-NK and the novel CAR-NK of the present invention co-cultured with tumor cells;

[0036] 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;

[0037] 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

[0038] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only 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 efforts are within the scope of protection of the present invention.

[0039] As used herein, "and / or" includes any and all combinations of one or more of the associated listed items.

[0040] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0041] 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.

[0042] In this specification, certain embodiments may be disclosed in a format that is within a range. It should be understood that this description of "within a range" is merely for convenience and brevity and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values within this range. For example, the description of a range of 1-6 should be considered to have specifically disclosed subranges 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.

[0043] Example 1

[0044] This example provides an example of a novel chimeric antibody receptor structure.

[0045] 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. Figure 1 shown.

[0046] Green fluorescent protein (GFP), a protein isolated from the jellyfish Aequorea Victoria, is composed of approximately 238 amino acids. It can be excited by light from blue to ultraviolet, emitting green fluorescence. This protein is widely used for cell labeling. This invention, for the first time, utilizes a fragment of this protein to enhance the function of CAR.

[0047] The CAR fragment in this 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 signaling domain.

[0048] It will be understood that the CAR fragment in this novel chimeric antibody receptor structure is only an example and not a limitation.

[0049] Example 2

[0050] This example provides an example of a method for constructing a novel chimeric antibody receptor structure (referred to as the novel CAR structure).

[0051] 1.1 Construction of novel chimeric antibody receptor structure and viral packaging.

[0052] A molecular cloning method was used to construct a lentiviral expression vector pWPXLD-CAR-P2A-X1 expressing a novel CAR structure. 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. The X1 fragment was connected via P2A, where X1 represents green fluorescent protein.

[0053] The structural sequences involved in this example are shown in Table 1.

[0054] Table 1

[0055]

[0056] 1.2 Packaging of the new CAR structure lentivirus.

[0057] Well-growing 293T cells were trypsinized and passaged at a density of 4 x 10^6 cells / dish. When the cells reached 80% confluence, they were transfected using calcium phosphate transfection with the CAR vector as the core plasmid and psPAX2 and pMD2.G as helper plasmids. Eight hours after transfection, the culture medium containing the calcium phosphate precipitate was discarded. 48 and 72 hours after transfection, the cell culture supernatant was collected as the viral stock solution and centrifuged at 2000 rpm for 10 minutes. The cell pellet was removed, and cellular debris was removed using a 0.22μm disposable syringe filter (PES membrane).

[0058] Example 3

[0059] Detection of the positive rate of the novel CAR structure synthesized in Example 2.

[0060] 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. The control group was a CAR without green fluorescent protein. Its structure was the same as the CAR shown in Example 2. The results are shown in FIG. Figure 2 shown.

[0061] The results showed that the positive expression rate of CAR could be increased after connection with green fluorescent protein.

[0062] Example 4

[0063] The tumor killing ability of the novel CAR structure synthesized in Example 2 was verified.

[0064] 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 cell types were co-cultured at an effector-target ratio of 5:1, and the killing effect of CAR-T cells was monitored.

[0065] Microscopically, CAR-T cells containing GFP short peptides can better gather tumor cells together and kill them ( Figure 3 ).

[0066] In addition, the killing effect of the killing curve was analyzed for 40 hours, 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 on, it can be seen that the CAR-peptide has a significantly higher ability to kill tumor cells than the control group CAR, and its killing efficiency is higher than the control group and the killing time is earlier than the control group.

[0067] The novel CAR-NK cells were further transduced with the lentivirus described in Example 2. SK-OV3 cells positive for the CAR target 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, and the killing effect of the CAR-T cells was monitored.

[0068] Analysis of the killing effect of the killing curve for 30 hours showed that the killing ability of CAR-peptide on tumor cells was higher than that of the control group CAR ( Figure 5 ).

[0069] Example 4

[0070] The CAR-T and CAR-NK of Example 3 were further verified.

[0071] The supernatants of CAR-T cells and CAR-NK cells were collected after 24 hours of co-culture with tumor cells, and IFN-γ, IL-2, and TNF-α in the supernatants were analyzed. IFN-γ and IL-2 in the supernatants increased significantly after co-culture of CAR-T cells with target cells ( 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.

[0072] Table 2 CAR-T cell detection results

[0073]

[0074] The results showed that green fluorescent egg, as an immunomodulatory factor, significantly increased the expression of IFN-γ and IL-2 in CAR-T cells compared with the control group.

[0075] Table 3 CAR-NK cell detection results

[0076]

[0077] The results showed that green fluorescent protein, as an immunomodulatory factor, significantly increased the expression of IFN-γ and TNF-α by CAR-NK cells compared to the control group, where CAR-NK cells barely expressed TNF-α.

[0078] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0079] 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 methods. The above-mentioned specific implementation methods are merely illustrative and not 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 protected by the present invention.

Claims

1. A CAR-T cell with anti-tumor effect, expressing 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 gene fragment of green fluorescent protein is shown in SEQ ID NO.1; the chimeric antigen receptor fragment includes 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; the nucleotide sequence of the chimeric antigen receptor fragment is shown in SEQ ID NO.3; the green fluorescent protein is used for immune activation of the chimeric antigen receptor; the novel chimeric antigen receptor is also connected to the EF1-α promoter sequence.

2. A CAR-NK cell with anti-tumor effect, expressing 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 gene fragment of green fluorescent protein is shown in SEQ ID NO.1; the chimeric antigen receptor fragment includes 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; the nucleotide sequence of the chimeric antigen receptor fragment is shown in SEQ ID NO.3; the green fluorescent protein is used for immune activation of the chimeric antigen receptor; the novel chimeric antigen receptor is also connected to the EF1-α promoter sequence.

3. A drug for treating solid tumors, characterized in that: Comprising the CAR-T cell according to claim 1.

4. A drug for treating solid tumors, characterized in that: Comprising the CAR-NK cell of claim 2.

5. A 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 a green fluorescent protein gene fragment, the nucleotide sequence of the green fluorescent protein gene fragment is shown in SEQ ID NO.1, and the nucleotide sequence of the chimeric antigen receptor fragment is shown in SEQ ID NO.

3.

6. The use according to claim 5, characterized in that The chimeric antigen receptor immune activator also contains a lentiviral expression vector.

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