Immune engineering cell for expressing continuous truncated functional region of GM-CSF and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to an immune engineering cell for expressing a continuous truncated functional region of a GM-CSF and application of the immune engineering cell. The invention firstly provides a CAR-NK cell for expressing a continuous truncated functional region of GM-CSF, and the CAR-NK cell expresses a novel chimeric antigen receptor structure; the chimeric antigen receptor structure comprises a continuous truncated functional domain fragment of a GM-CSF protein and a chimeric antigen receptor; the continuous truncated functional domain fragment of the GM-CSF protein comprises an A helix fragment, an exon 1 fragment and a C helix fragment. The invention also provides a CAR-T cell which also expresses the continuous truncated functional region of the GM-CSF.

Description

[0001] Priority application This application claims the priority of the Chinese invention patent application [CN2023115954083] filed on November 24, 2023 "A CAR-T comprising the 5th truncation region of GM-CSF and its preparation method and application", and the priority of the Chinese invention patent application [CN2023115817245] filed on November 24, 2023 "A CAR-T comprising the 2nd truncation region of GM-CSF and its preparation method and application". The two priority invention patent applications are incorporated by reference in their entirety. Technical Field

[0002] The invention belongs to the field of biotechnology, and in particular relates to immune engineering cells expressing continuously truncated functional regions of GM-CSF and applications 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] Chimeric antigen receptor (CAR) is an artificial receptor molecule manufactured by genetic engineering technology. It can give immune effector cells (such as T cells and NK cells) specificity for a target antigen epitope, thereby enhancing the function of lymphocytes to recognize antigen signals and activate. The first-generation CAR has only CD3 ξ (containing 3 ITAMs) and FcRγ (containing 2 ITAMs) signal transduction domains in the cell, which can only provide the first signal for T cell activation. Early clinical trials of the first-generation CAR showed that the first-generation CAR-T could not effectively maintain the proliferation and activation of T cells, and did not achieve satisfactory results, indicating that the first-generation CAR-T cells may lack sufficient activation signals to maintain T cell proliferation and effective anti-tumor effects. Researchers developed the second-generation CAR-T based on the intracellular signal sequence of the first-generation CAR-T. The second-generation CAR-T cells showed enhanced in vivo expansion and persistence, and their effects have 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 for 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. Therefore, this field still needs technological innovation.

[0006] 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. 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. The anti-cancer effect of immune cells can be significantly improved by expressing chimeric antigen receptors (CARs) on the surface of NK cells. At present, the scientific community believes that CAR-NK cells have some significant advantages over CAR-T cells. However, the preparation of CAR-NK cells with tumor-killing efficacy is somewhat difficult, mainly because NK cells have natural antiviral functions and their genetic modification is difficult.

[0007] The patent with publication number CN114934071A and invention name "A CAR vector expressing an immunomodulatory factor and its application" discloses a CAR-T cell expressing a chimeric antigen receptor and an immunomodulatory factor transduced by a virus, and the immunomodulatory factor is the full length of granulocyte-macrophage colony stimulating factor (GM-CSF). NK cell activation receptors include natural cytotoxicity receptors (NCRs), such as NKG2D, CD16 (FcgRIIIa), FasL and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as well as co-stimulatory receptors, such as LFA-1, CD244 (2B4) and CD137 (41BB), which are quite different from the intracellular signal activation domain of CAR-T cells. Therefore, the traditional CAR molecule design that works on CAR-T may not necessarily play a good activation role in NK cells.

[0008] Therefore, it is necessary to improve the existing technology. Summary of the invention

[0009] In view of this, the object of the present invention is to provide an immunoengineered cell expressing the first continuous truncated functional region of GM-CSF and its application. The present invention specifically adopts the following technical solutions.

[0010] One aspect of the present invention is to provide a CAR-NK cell expressing the first continuously truncated functional region of GM-CSF.

[0011] A CAR-NK cell expressing a continuously truncated functional region of GM-CSF, wherein the CAR-NK cell expresses a novel chimeric antigen receptor structure; the chimeric antigen receptor structure comprises a continuously truncated functional region fragment of a GM-CSF protein and a chimeric antigen receptor; the continuously truncated functional region fragment of the GM-CSF protein comprises an A helix fragment, an exon 1 fragment and a Chelix fragment; the continuously truncated functional region fragment of the GM-CSF protein is connected to the chimeric antigen receptor via a P2A sequence; the chimeric antigen receptor comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB costimulatory signal region and a human CD3 ζ signal domain.

[0012] Furthermore, the nucleotide sequence of the continuously truncated functional region fragments of the GM-CSF protein is shown in SEQ ID NO.1.

[0013] Furthermore, the novel chimeric antigen receptor structure is also connected to an EF1-α promoter sequence.

[0014] Furthermore, the nucleotide sequence of the chimeric antigen receptor expressed by the CAR-NK cell is shown in SEQ ID NO.3.

[0015] Another aspect of the present invention is to provide a CAR-T cell expressing the first continuously truncated functional region of GM-CSF.

[0016] A CAR-T cell expressing a continuously truncated functional region of GM-CSF, wherein the CAR-T cell expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises a continuously truncated functional region fragment of a GM-CSF protein and a chimeric antigen receptor; the continuously truncated functional region fragment of the GM-CSF protein comprises an A helix fragment, an exon 1 fragment and a Chelix fragment; the continuously truncated functional region fragment of the GM-CSF protein is connected to the chimeric antigen receptor via a P2A sequence; the chimeric antigen receptor comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB costimulatory signal region and a human CD3 ζ signal domain.

[0017] Furthermore, the nucleotide sequence of the continuously truncated functional region fragments of the GM-CSF protein is shown in SEQ ID NO.1.

[0018] Furthermore, the novel chimeric antigen receptor structure is also connected to an EF1-α promoter sequence.

[0019] Furthermore, the nucleotide sequence of the chimeric antigen receptor expressed by the CAR-T cell is shown in SEQ ID NO.3.

[0020] The present invention also provides a lentiviral vector of a CAR expression vector, which includes a nucleic acid encoding a chimeric antigen receptor and a nucleic acid encoding a continuously truncated functional region of GM-CSF; the nucleic acid encoding the chimeric antigen receptor and the nucleic acid encoding the continuously truncated functional region of GM-CSF are connected through a sequence encoding P2A; the fragments of the continuously truncated functional region of GM-CSF include an A helix fragment, an exon 1 fragment and a Chelix fragment; the chimeric antigen receptor 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 CD3 ζ signal domain; the lentivirus includes pWPXLD, psPAX2 and pMD2.G.

[0021] The antigen binding domain in the CAR provided by the present invention can target CD19, BCMA, HER2, Claudin18.2, Mesothelin, GPC3, GD2, etc.

[0022] The use of the above-mentioned CAR-NK cells in the preparation of TNF-α promoters.

[0023] The CAR-NK cells can promote the expression of TNF-α in tumor tissues or cells.

[0024] The use of the above-mentioned CAR-NK cells in the preparation of IFN-γ promoters.

[0025] The CAR-NK cells can promote the expression of IFN-γ in tumor tissues or cells.

[0026] The use of the above-mentioned CAR-T cells in the preparation of drugs for treating solid tumors.

[0027] Furthermore, the tumor includes hematological tumor, liver cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, ovarian cancer, glioma, pancreatic cancer, sarcoma or glioblastoma.

[0028] Beneficial technical effects: On the one hand, the present invention constructs an immune engineering cell expressing a continuous truncated functional region of GM-CSF, which expresses a novel chimeric antigen receptor structure. Compared with the GM-CSF full-length protein, the novel chimeric antigen receptor structure of the present invention only contains part of the continuous truncated functional region of the GM-CSF full-length protein, which reduces the sequence length of the overexpressed gene, but does not affect the expression and function of CAR. On the contrary, since the continuous truncated functional region does not contain a signal peptide, it can function in the cell, enhance the expression of CAR and improve the proliferation ability of CAR-T cells.

[0029] Moreover, the experiment of the present invention further confirmed that under the condition that all the functional regions are continuously truncated, different numbers of continuously truncated functional regions will also lead to different technical effects. Specifically, the proliferation ability of the CAR-T cells (CAR-X5-T) transformed with three continuously truncated functional regions expressing GM-CSF of the present invention is significantly better than that of conventional CAR-T cells and CAR-T cells (CAR-GM-T) prepared by full-length GM-CSF, and comparative analysis found that the proliferation effect of CAR-X5-T cells is significantly better than that of CAR-T cells (CAR-X2-T) transformed with two continuously truncated functional regions expressing GM-CSF. It shows that under the condition that all the functional regions are continuously truncated, further shortening the number of continuously truncated functional regions may not necessarily have a better effect on promoting the proliferation of CAR-T cells. Obviously, the CAR-X5-T cells of the present invention have higher yields and lower production costs when used for industrial production, and have greater industrial promotion value.

[0030] On the other hand, the present invention experiments confirmed that CAR-NK cells (CAR-X5-NK) expressing the continuous truncated functional regions of GM-CSF have a strong cytokine secretion ability, and the TNF-α and IFN-γ secreted by them are significantly higher than those of conventional CAR-NK cells. Therefore, the CAR-X5-NK of the present invention is suitable for the preparation of TNF-α promoters and IFN-γ promoters, and has great industrial production value.

[0031] In summary, due to the differences between NK cell modification and T cell modification, the CAR structure expressed and functioning on CAR-T cells does not necessarily have the same effect on CAR-NK cells. In addition, unlike the conjecture of those skilled in the art, the smaller the length of the expression factor, the more enhanced the function of CAR, which requires further experimental verification. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] 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.

[0033] Figure 1 This is a diagram of the construction of the novel CAR structure expression vector of the present invention; Figure 2 It is a schematic diagram of the structure of the functional region of the full-length GM-CSF protein; Figure 3This is a result diagram of the CAR positivity detection of CAR-X5-T cells constructed in one of the embodiments of the present invention; Figure 4 This is a result diagram of the proliferation of CAR-X5-T cells constructed in one of the embodiments of the present invention; Figure 5 This is a graph showing the proliferation of CAR-X2-T cells in the control group constructed in one of the embodiments of the present invention; Figure 6 This is a killing curve of tumor cells by CAR-X5-T cells constructed in one embodiment of the present invention; Figure 7 The figure shows the detection results of TNF-α and IFN-γ after co-culture of CAR-X5-NK cells and tumor cells when the effector-target cell ratio is 5:1; Figure 8 This is a killing curve of tumor cells by CAR-X5-NK cells constructed in one of the embodiments of the present invention. DETAILED DESCRIPTION

[0034] 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.

[0035] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0036] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0037] 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.

[0038] 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.

[0039] Definition of noun: The "novel chimeric antigen receptor structure" described in the present invention refers to a chimeric antigen receptor (CAR) and a continuous truncated functional region fragment (X5) of the GM-CSF protein connected to the chimeric antigen receptor by P2A. The chimeric antigen receptor 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 CD3 ζ signal domain.

[0040] The "GM-CSF truncated region" of the present invention refers to a truncated region obtained by modifying the functional region fragments of the full-length GM-CSF protein by truncating or deleting the functional region fragments; wherein the functional region fragments include signal peptide fragments, Ahelix fragments, exon 1 fragments, Chelix fragments and exon 2 fragments. The full-length structure of the GM-CSF protein is as follows Figure 2 shown.

[0041] Based on the above transformation methods, the full-length GM-CSF protein was prepared into 5 short peptides containing different truncated fragments, including: GM-CSF first truncation region: contains only the A helix fragment region of GM-CSF.

[0042] GM-CSF second truncation region: contains the exon 1 fragment region and the Chelix fragment region of GM-CSF.

[0043] GM-CSF 3rd truncation region: only contains the exon 2 fragment region of GM-CSF.

[0044] GM-CSF 4th truncation region: includes the A helix, Chelix and exon 2 fragments of GM-CSF.

[0045] GM-CSF 5th truncation region: contains the A helix, exon 1 and Chelix fragment regions of GM-CSF.

[0046] On the one hand, the purpose of the present invention is to explore the improvement of CAR function by truncating the full-length GM-CSF protein.

[0047] Another invention is to explore the effects of different numbers of functional regions on CAR function under the premise of continuous truncation of functional regions.

[0048] In the following examples, for the sake of distinction, a short peptide comprising three consecutive truncated functional regions is referred to as the first consecutive truncated functional region, represented by X5; a short peptide comprising two consecutive truncated functional regions is referred to as the second consecutive truncated functional region, represented by X2.

[0049] The sequence information involved in the present invention is shown in Table 1.

[0050] Table 1 Example 1 Construction and viral packaging of a novel CAR expression vector.

[0051] 1.1 Construction of lentiviral expression vector of novel CAR vector.

[0052] The lentiviral expression vector pWPXLD-CAR-P2A-X5 expressing the novel CAR was constructed by molecular cloning. The CAR fragment contained CD8 signal peptide, antigen recognition region (SCFV), hinge region (hinge), human CD8 transmembrane region (TM), human 4-1BB costimulatory signal region, human CD3 ζ signal domain, P2A, and fragment (X5), such as Figure 1 shown.

[0053] 1.2 Packaging of novel CAR lentivirus.

[0054] Take 293T cells with good growth status, digest the cells with trypsin, and then add 4×10 6 The cells were passaged at a density of 100 cells / dish. When the cell confluence reached 80%, the CAR vector was used as the core plasmid, and psPAX2 and pMD2.G were used as auxiliary plasmids, using calcium phosphate transfection technology for transfection. Eight hours after transfection, the culture medium containing calcium phosphate precipitation was discarded. After 48 and 72 hours of transfection, the cell culture supernatant was collected as the virus stock solution, centrifuged at 2000 rpm for 10 minutes, the cell precipitate was removed, and a 0.22μm disposable syringe filter (PES membrane) was used to remove cell debris.

[0055] Example 2 CAR positivity rate detection.

[0056] The lentivirus in Example 1 was used to transduce T cells to obtain novel CAR-X5-T cells, and 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. Conventional CAR is a traditional second-generation CAR-T cell, CAR-GM is a CAR-T cell containing the full-length GM, and CAR-X5 is a CAR-T cell containing the fragment X5.

[0057] The results are as follows Figure 3 The CAR-X5-T cells showed a positive rate of 61.83%, which was higher than that of conventional CAR-T cells and CAR-GM-T cells, indicating that only part of the GM-CSF functional region was contained and the functional region was a continuous truncated region, which could enhance the expression of CAR.

[0058] Example 3 This example detects the proliferation ability of the prepared CAR-X5-T cells.

[0059] The same number of T cells were transduced using the lentivirus in Example 1 to obtain novel CAR-X5-T cells (experimental group). After 3 days, the number of CAR-X5-T cells was detected using a cell counter and the expansion multiple was calculated. The results are as follows: Figure 4 As shown, the proliferation ability of CAR-X5-T cells is significantly better than that of conventional CAR-T cells and CAR-GM-T cells, indicating that reducing the length of the GM-CSF functional region improves the proliferation ability of CAR-T cells, suggesting that CAR-T cells modified with the "first continuous truncated functional region" containing GM-CSF have better proliferation effect.

[0060] Further, using a similar method to Example 1, lentivirus was used to transduce T cells to obtain CAR-X2-T cells (control group), and their proliferation was regularly detected. The results are as follows: Figure 5 As shown in the figure, through comparative analysis, it was found that the proliferation ability of CAR-X5-T cells was significantly better than that of CAR-X2-T cells, suggesting that under the premise that both are continuously truncated functional regions, further shortening the number of continuously truncated functional regions of GM-CSF (i.e., the "second continuously truncated functional region") may not necessarily have a better effect on promoting the proliferation of CAR-T cells.

[0061] The above experimental results show that containing a specific number of continuously truncated functional regions of GM-CSF protein (i.e., the "first continuously truncated functional region") can not only promote the expression of CAR, but also enhance the proliferation ability of CAR-T cells, and its proliferation ability is significantly stronger than that of CAR-T prepared with full-length GM-CSF and CAR-T expressing the "second continuously truncated functional region" of GM-CSF.

[0062] In the CAR-X2-T cells involved in this embodiment, the nucleotide sequence of X2 is as follows.

[0063] Table 2 Example 4 This example provides verification of CAR-X5-T cells killing tumor cells.

[0064] CAR target-positive SK-OV3 cells (human ovarian cancer cells) were used as target cells, and the CAR-X5-T cells obtained in Example 3 were used as effector cells. The two cells were co-cultured at an effector-target ratio of 5:1, and the killing of SK-OV3 cells by CAR-X5-T cells was monitored.

[0065] The results are as follows Figure 6 As shown in the figure, it can be seen that the killing ability of CAR-X5-T cells on tumor cells is significantly higher than that of conventional CAR-T cells starting from about 10 hours. Analysis of the killing effect of the killing curve for 40 hours generally shows that the killing efficiency of CAR-X5-T cells is similar to that of CAR-GM-T cells, but significantly higher than that of conventional CAR-T cells, and the killing effect is also earlier than that of conventional CAR-T cells.

[0066] The above experimental results show that appropriately reducing the length of the GM-CSF functional region can enhance the proliferation ability of CAR-T cells without significantly weakening its killing effect on tumor cells.

[0067] Example 5 This example detects the cytokine secretion ability of CAR-X5-NK cells.

[0068] The lentivirus in Example 1 was used to transduce NK cells to obtain novel CAR-X5-NK cells. The supernatants of CAR-NK cells and tumor cells were collected after 24 hours of co-culture, and IFN-γ and TNF-α in the supernatants were analyzed. After CAR-X5-NK cells were co-cultured with target cells, IFN-γ and TNF-α in the supernatants increased significantly ( Figure 7 ), the specific results are shown in Table 2.

[0069] Table 3 Detection results of cytokines secreted by CAR-NK cells.

[0070] The results showed that compared with conventional CAR, CAR-NK cells modified with the "first continuous truncated functional region" of GM-CSF secreted 76.9 times more TNF-α and 1.9 times more IFN-γ than conventional CAR-NK cells, suggesting that CAR-NK cells expressing an appropriate number of continuous truncated functional regions of GM-CSF protein can significantly enhance the cytokine secretion ability of CAR-NK cells.

[0071] Example 6 This example further demonstrates the ability of CAR-X5-NK cells to kill tumor cells based on Example 5.

[0072] SK-OV3 cells with positive CAR target were used as target cells, and CAR-X5-NK cells in Example 5 were used as effector cells. The two cells were co-cultured at an effector-target ratio of 5:1, and the killing of SK-OV3 cells by CAR-X5-NK cells was monitored. The results are shown in Figure 8 As shown, the killing efficacy of the killing curve was analyzed for 30 hours, which generally showed that the killing ability of CAR-X5-NK cells against tumor cells was slightly weaker than that of conventional CAR-NK cells.

[0073] The above results indicate that although the CAR-NK cells modified with an appropriate number of consecutive truncated functional regions of the GM-CSF protein (i.e., the "first consecutive truncated functional region") have stronger cytokine secretion ability than conventional CAR-NK cells, their tumor cell killing effect is not necessarily better than that of conventional CAR-NK cells.

[0074] 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.

[0075] 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 CAR-NK cell expressing a continuously truncated functional region of GM-CSF, characterized in that: The CAR-NK cell expresses a novel chimeric antigen receptor structure; the chimeric antigen receptor structure comprises a continuously truncated functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the continuously truncated functional region fragment of the GM-CSF protein comprises an A helix fragment, an exon 1 fragment and a Chelix fragment; the continuously truncated functional region fragment of the GM-CSF protein is connected to the chimeric antigen receptor via a P2A sequence; the chimeric antigen receptor 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 CD3 ζ signal domain.

2. The CAR-NK cell according to claim 1, wherein The nucleotide sequence of the continuously truncated functional region fragment of the GM-CSF protein is shown in SEQ ID NO.

1.

3. The CAR-NK cell according to claim 1, wherein The novel chimeric antigen receptor structure is also connected to the EF1-α promoter sequence.

4. The CAR-NK cell according to claim 1, wherein The nucleotide sequence of the chimeric antigen receptor expressed by the CAR-NK cell is shown in SEQ ID NO.

3.

5. A CAR-T cell expressing GM-CSF continuously truncated functional regions, characterized in that: The CAR-T cell expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises a continuously truncated functional region fragment of a GM-CSF protein and a chimeric antigen receptor; the continuously truncated functional region fragment of the GM-CSF protein comprises an A helix fragment, an exon 1 fragment and a Chelix fragment; the continuously truncated functional region fragment of the GM-CSF protein is connected to the chimeric antigen receptor via a P2A sequence; the chimeric antigen receptor 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 CD3 ζ signal domain.

6. The CAR-T cell according to claim 5, wherein The nucleotide sequence of the continuously truncated functional region fragment of the GM-CSF protein is shown in SEQ ID NO.

1.

7. The CAR-T cell according to claim 5, characterized in that The novel chimeric antigen receptor structure is also connected to the EF1-α promoter sequence.

8. The CAR-T cell according to claim 5, wherein The nucleotide sequence of the chimeric antigen receptor expressed by the CAR-T cell is shown in SEQ ID NO.

3.

9. Use of the CAR-NK cells according to any one of claims 1 to 4 in the preparation of a TNF-α promoter; or use of the CAR-NK cells according to any one of claims 1 to 4 in the preparation of an IFN-γ promoter.

10. Use of the CAR-T cell according to any one of claims 5 to 8 in the preparation of a drug for treating solid tumors.

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