Immunologically engineered cells expressing noncontiguous truncated functional domains of gm-csf and uses thereof

Abstract

The application belongs to the technical field of biology and specifically relates to immune engineering cells expressing non-continuous truncated functional regions of GM-CSF and application. The application first provides a CAR-NK cell expressing non-continuous truncated functional regions of GM-CSF, which expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises a non-continuous truncated functional region fragment of GM-CSF protein and a chimeric antigen receptor; the non-continuous truncated functional region fragment of GM-CSF protein comprises an A helix fragment, a C helix fragment and an exon 2 fragment. The application also provides a CAR-T cell also expressing non-continuous truncated functional regions of GM-CSF.

Description

[0001] Priority application

[0002] This application claims priority to Chinese invention patent application No. CN2023115953911, filed November 24, 2023, entitled “A CAR-T containing the fourth truncated region of GM-CSF, a preparation method and application thereof”, and Chinese invention patent application No. CN2023115954083, filed November 24, 2023, entitled “A CAR-T containing the fifth truncated region of GM-CSF, a preparation method and application thereof”, both of which are incorporated herein by reference in their entirety. Technical Field

[0003] This invention belongs to the field of biotechnology, specifically relating to immune engineered cells expressing discontinuously truncated functional regions of GM-CSF and their applications. Background Technology

[0004] For tumors, traditional surgical removal, chemotherapy, and radiotherapy are no longer sufficient for effective monitoring and treatment, necessitating effective alternative therapies to overcome this challenge. In recent years, the emergence of immunotherapy has brought revolutionary progress to the field of tumor treatment, with remarkable achievements in areas such as immune checkpoint inhibitors, bispecific antibodies, and CAR-T cell therapy.

[0005] Chimeric antigen receptors (CARs) are artificial receptor molecules manufactured using genetic engineering technology. They can endow immune effector cells (such as T cells and NK cells) with specificity for a target antigen epitope, thereby enhancing the function of lymphocytes in recognizing antigen signals and activating. First-generation CARs only contain CD3ξ (containing 3 ITAMs) and FcRγ (containing 2 ITAMs) signaling domains, providing only the first signal for T cell activation. Early clinical trials of first-generation CARs showed that they could not effectively maintain T cell proliferation and activation, failing to achieve satisfactory results, indicating that first-generation CAR-T cells may lack sufficient activation signals to maintain T cell proliferation and effective anti-tumor effects. Based on first-generation CAR-T, second-generation CAR-T was developed. Second-generation CAR-T cells have shown enhanced in vivo expansion and persistence, and their efficacy has been confirmed in clinical trials.

[0006] Since then, CAR-T related technologies have been continuously innovated, but of the 939 immunotherapy trials initiated since 1993, only about half have targeted solid tumors. However, solid tumors account for 90% of global cancer incidence. Unfortunately, compared to the good efficacy shown in hematologic malignancies, most reported efficacy of CAR-T cell therapy for solid tumors has consistently failed to achieve significant breakthroughs at certain key stages. Therefore, technological innovation is still needed in this field.

[0007] NK cells are important members of the innate immune system, mainly found in blood and lymphatic organs. They do not require pre-sensitization and are not restricted by the major histocompatibility complex (MHC), enabling them to rapidly and directly kill target cells and exhibiting broad-spectrum anti-tumor activity. NK cells kill target cells in various ways. Once the balance between inhibitory and activating signals within NK cells shifts towards activation, NK cells can form synapses with target cells, releasing effector granules to lyse the target cells and producing effector cytokines. Expressing chimeric antigen receptors (CARs) on the surface of NK cells can significantly enhance the anti-cancer effect of immune cells. Currently, the scientific community believes that CAR-NK cells have some significant advantages compared to CAR-T cells. However, the preparation of CAR-NK cells with tumor-killing efficacy is challenging, mainly because the genetic modification of NK cells, due to their natural antiviral function, is difficult.

[0008] The patent, CN114934071A, entitled "A CAR Vector Expressing an Immunomodulatory Factor and Its Application," discloses a CAR-T cell expressing a virally transduced chimeric antigen receptor and an immunomodulatory factor, the full-length granulocyte-macrophage colony-stimulating factor (GM-CSF). NK cell activation receptors include natural cytotoxic receptors (NCRs) such as NKG2D, CD16 (FcgRIIIa), FasL, and tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL), as well as co-stimulatory receptors such as LFA-1, CD244 (2B4), and CD137 (41BB). However, the intracellular signaling activation domains of CAR-T cells are mainly 41BB, CD28, and CD3ζ. Therefore, traditional CAR molecule designs that are effective for CAR-T cells may not necessarily exert good activation effects in NK cells.

[0009] In conclusion, it is necessary to make improvements to the existing technology. Summary of the Invention

[0010] In view of this, the purpose of the present invention is to provide immune-engineered cells expressing discontinuously truncated functional regions of GM-CSF, their preparation methods and applications, and the specific technical solutions are as follows.

[0011] One aspect of the present invention is to provide a CAR-NK cell expressing a discontinuously truncated functional region of GM-CSF.

[0012] A CAR-NK cell expressing a discontinuously truncated functional region of GM-CSF, wherein the CAR-NK cell expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises a discontinuously truncated functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the discontinuously truncated functional region fragment of the GM-CSF protein includes an A helix fragment, a C helix fragment, and an exon 2 fragment; the discontinuously truncated functional region fragment of the GM-CSF protein is linked to the chimeric antigen receptor via a P2A sequence; 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 signaling region, and a human CD3ζ signaling domain.

[0013] Furthermore, the nucleotide sequence of the discontinuous truncated functional region fragment of the GM-CSF protein is shown in SEQ ID NO.1.

[0014] Furthermore, the novel chimeric antigen receptor structure is also linked to an EF1-α promoter sequence.

[0015] Furthermore, the nucleotide sequence of the chimeric antigen receptor expressed by the CAR-NK cells is shown in SEQ ID NO.3.

[0016] Another aspect of the present invention is to provide a CAR-T cell expressing a discontinuously truncated functional region of GM-CSF.

[0017] A CAR-T cell expressing a discontinuously 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 discontinuously truncated functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the discontinuously truncated functional region fragment of the GM-CSF protein includes an A helix fragment, a C helix fragment, and an exon 2 fragment; the discontinuously truncated functional region fragment of the GM-CSF protein is linked to the chimeric antigen receptor via a P2A sequence; 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 signaling region, and a human CD3ζ signaling domain.

[0018] Furthermore, the nucleotide sequence of the discontinuous truncated functional region fragment of the GM-CSF protein is shown in SEQ ID NO.1.

[0019] Furthermore, the novel chimeric antigen receptor structure is also linked to an EF1-α promoter sequence.

[0020] Furthermore, the nucleotide sequence of the chimeric antigen receptor expressed by the CAR-T cells is shown in SEQ ID NO.3.

[0021] The application of the above-mentioned CAR-NK cells in the preparation of drugs for treating solid tumors. The application of the above-mentioned CAR-T cells in the preparation of drugs for treating solid tumors.

[0022] Compared with CAR-T cells, CAR-NK cells have several 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 broad-spectrum and highly efficient tumor-killing activity. (3) Allogeneic infusion therapy can be achieved.

[0023] The aforementioned CAR-NK cells or CAR-T cells can enhance their killing effect on tumor cells by expressing the discontinuous truncated functional region of GM-CSF.

[0024] The antigen-binding domain in the CAR provided by this invention can target CD19, BCMA, HER2, Claudin18.2, Mesothelin, GPC3, GD2, etc.

[0025] Furthermore, the tumors include hematologic malignancies, liver cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, ovarian cancer, glioma, pancreatic cancer, sarcoma, or glioblastoma.

[0026] The present invention may also include a method for preparing the above-mentioned CAR, or a method for preparing the above-mentioned CAR-T or CAR-NK.

[0027] Beneficial technical effects:

[0028] On one hand, this invention constructs immunoengineered cells expressing a discontinuously truncated functional region of GM-CSF, which expresses a novel chimeric antigen receptor structure. Compared to the full-length GM-CSF protein, the novel chimeric antigen receptor structure of this invention only contains a partially discontinuously truncated functional region of the full-length GM-CSF protein, reducing the sequence length of the overexpressed gene without affecting CAR expression and function. On the contrary, since this discontinuously truncated functional region does not contain a signal peptide, it can function intracellularly, enhancing the proliferation capacity of CAR-T cells. The proliferation capacity of the CAR-X4-T cells of this invention is significantly superior to that of conventional CAR-T cells and CAR-GM-T cells. Obviously, the CAR-X4-T cells of this invention have higher yields and lower production costs when used for industrial production, and have greater industrial application value.

[0029] On the other hand, the experiments of this invention have confirmed that, under the premise of containing the same number of functional regions of the full-length GM-CSF protein, different sequence modification methods lead to different technical effects. This invention has demonstrated that CAR-NK cells modified with discontinuously truncated functional regions expressing GM-CSF (CAR-X4-NK) have a higher tumor cell killing efficiency than CAR-NK cells modified with continuously truncated functional regions expressing GM-CSF (CAR-X5-NK), and their overall cytokine secretion capacity is stronger, resulting in a more significant enhancement of the CAR effect. Specifically, the CAR-X4-NK cells of this invention exhibit tumor cell killing activity significantly earlier than conventional CAR-NK cells and CAR-X5-NK cells, while the overall killing effect of CAR-X5-NK cells is weaker than that of conventional CAR-NK cells. Furthermore, the TNF-α and IFN-γ secreted by CAR-X4-NK cells and CAR-X5-NK cells are significantly higher than those of conventional CAR-NK cells, and overall, CAR-X4-NK cells have a better cytokine secretion capacity than CAR-X5-NK cells. Therefore, the CAR-X4-NK cells of the present invention can serve as an adjuvant product for cell therapy and play a greater role in the preparation of anti-tumor therapeutic drugs.

[0030] Finally, due to the differences between NK cell modification and T cell modification, CAR structures that are expressed and function on CAR-T cells may not necessarily have the same function on CAR-NK cells, which requires experimental verification. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0032] Figure 1 This is a construction diagram of the novel CAR structure expression vector of the present invention;

[0033] Figure 2 This is a schematic diagram of the functional region of the full-length GM-CSF protein;

[0034] Figure 3 This is a graph showing the CAR positivity rate detection results of CAR-X4-T cells constructed in one embodiment of the present invention;

[0035] Figure 4This is a graph showing the proliferation results of CAR-X4-T cells constructed in one embodiment of the present invention;

[0036] Figure 5 This is a curve showing the killing effect of CAR-X4-T cells on tumor cells constructed in one embodiment of the present invention;

[0037] Figure 6 This is a curve showing the killing effect of CAR-X4-NK cells on tumor cells constructed in one embodiment of the present invention;

[0038] Figure 7 This is a curve showing the killing effect of CAR-X5-NK cells on tumor cells in a control group constructed in one embodiment of the present invention.

[0039] Figure 8 The image shows the TNF-α secretion results of CAR-X4-NK cells and control CAR-X5-NK cells after co-culturing with tumor cells when the effector-target cell ratio is 5:1 (A: TNF-α secretion results of CAR-X4-NK cells, B: TNF-α secretion results of CAR-X5-NK cells).

[0040] Figure 9 The image shows the IFN-γ secretion results of CAR-X4-NK cells and control group CAR-X5-NK cells after co-culturing with tumor cells when the effector-target cell ratio is 5:1 (A: IFN-γ secretion results of CAR-X4-NK cells; B: IFN-γ secretion results of CAR-X5-NK cells). Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0042] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0043] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0044] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0045] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having 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., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0046] Definition of noun:

[0047] The "novel chimeric antigen receptor structure" described in this invention refers to a chimeric antigen receptor (CAR) and a discontinuous truncated functional region fragment (X4) of the GM-CSF protein linked via P2A outside the chimeric antigen receptor. 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 signaling region, and a human CD3ζ signaling domain.

[0048] The "GM-CSF truncated region" described in this invention refers to a truncated region obtained by modifying the full-length GM-CSF protein by cutting off and deleting functional region fragments; wherein, the functional region fragments include the signal peptid fragment, the A helix fragment, the exon 1 fragment, the C helix fragment, and the exon 2 fragment. The full-length structure of the GM-CSF protein is as follows. Figure 2 As shown.

[0049] Based on the above modification methods, the full-length GM-CSF protein was prepared into 5 short peptides containing different truncated fragments, specifically including:

[0050] GM-CSF 1st truncated region: A helix fragment region containing only GM-CSF, denoted by X1.

[0051] GM-CSF 2nd truncated region: Contains the exon 1 fragment region and the C helix fragment region of GM-CSF, denoted as X2.

[0052] GM-CSF 3rd truncated region: The region containing only the exon 2 fragment of GM-CSF, denoted as X3.

[0053] GM-CSF 4th truncated region: Contains the A helix, C helix and exon 2 fragment regions of GM-CSF, denoted as X4.

[0054] GM-CSF 5th truncated region: Contains the A helix, exon 1 and C helix fragment regions of GM-CSF, denoted as X5.

[0055] On the one hand, the purpose of this invention is to explore how truncating the full-length GM-CSF protein can improve CAR function.

[0056] Another invention aims to explore the impact of including continuously truncated functional areas and including non-continuously truncated functional areas on CAR functionality, provided that the same number of functional areas are included.

[0057] The “continuous truncated functional area” in this invention refers to the 5th truncated area, denoted by X5; the “non-continuous truncated functional area” in this invention refers to the 4th truncated area, denoted by X4.

[0058] The sequence information involved in this invention is shown in Table 1.

[0059] Table 1

[0060]

[0061] Example 1

[0062] Construction of novel CAR expression vectors and viral packaging.

[0063] 1.1 Construction of lentiviral expression vectors for novel CAR vectors.

[0064] A novel lentiviral expression vector, pWPXLD-CAR-P2A-X4, was constructed using molecular cloning methods. This expression vector contains a CD8 signal peptide, a single-chain antibody (SCFV), a hinge region, a human CD8 transmembrane region (TM), a human 4-1BB co-stimulatory signaling region, a human CD3 ζ signaling domain, P2A, and the X4 fragment. Figure 1 As shown.

[0065] 1.2 Packaging of novel CAR lentiviruses

[0066] Take 293T cells in good growth condition, digest the cells with trypsin, and then... 6The cells were passaged at a density of cells / plate until 80% confluence was achieved. Transfection was then performed using a CAR vector as the core plasmid and psPAX2 and pMD2.G as helper plasmids via calcium phosphate transfection. Eight hours after transfection, the medium containing the calcium phosphate precipitate was discarded. The cell culture supernatant was collected at 48 and 72 hours after transfection as the viral stock solution. The cells were centrifuged at 2000 rpm for 10 minutes to remove the cell pellet, and cell debris was removed using a 0.22 μm disposable syringe filter (PES membrane).

[0067] Example 2

[0068] CAR positivity rate detection.

[0069] Novel CAR-X4-T cells were obtained by transducing T cells with the lentivirus described in Example 1. The CAR positivity rate was detected using a His-tagged target protein as the primary antibody and a His antibody conjugated with fluorescein APC as the secondary antibody. Conventional CAR refers to traditional second-generation CAR-T, CAR-GM refers to a CAR containing the full-length GM fragment, and CAR-X4 refers to a CAR containing the X4 fragment. Results are as follows: Figure 3 As shown, the positivity rate of CAR in CAR-X4-T cells was 56.75%, which was comparable to that of conventional CAR and CAR-GM, indicating that it only contained a portion of the GM-CSF functional region and that the functional region was a non-contiguous truncated region, which would not affect CAR expression.

[0070] Example 3

[0071] This embodiment tests the proliferation ability of CAR-X4-T cells synthesized in Example 1.

[0072] The same number of T cells were transduced using the lentivirus from Example 1 to obtain novel CAR-X4-T cells. Three days later, the number of CAR-X4-T cells was detected using a cell counter, and the fold increase was calculated. The results are as follows: Figure 4 As shown, the proliferation capacity of CAR-X4-T cells is significantly better than that of conventional CAR-T cells and CAR-GM-T cells, demonstrating that reducing the length of GM-CSF enhances cell proliferation.

[0073] Example 4

[0074] This embodiment verifies the killing of tumor cells by the CAR-X4-T cells synthesized in Example 1.

[0075] CAR-target positive SK-OV3 cells (human ovarian cancer cells) were used as target cells, and CAR-X4-T cells obtained in Example 4 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-X4-T cells on SK-OV3 cells was monitored.

[0076] The results are as follows Figure 5 As shown, from approximately 5 hours onwards, the tumor-killing ability of CAR-X4-T cells is significantly higher than that of conventional CAR. Analysis of the killing curve at 40 hours reveals that the tumor-killing efficacy of CAR-X4-T cells is comparable to that of CAR-GM-T cells, and higher than that of conventional CAR.

[0077] The above experimental results show that reducing the length of GM-CSF can enhance the proliferation capacity of CAR-T cells without affecting their tumor-killing efficacy, which is comparable to CAR-T cells prepared from full-length GM-CSF.

[0078] Example 5

[0079] This embodiment provides verification of CAR-X4-NK cells killing tumor cells.

[0080] Using a method similar to that in Example 1, NK cells were transduced with lentivirus to obtain CAR-X4-NK cells (experimental group) and CAR-X5-NK cells (control group), respectively. The CAR-X4-NK and CAR-X5-NK cells have the same CAR structure.

[0081] CAR-target-positive SK-OV3 cells (human ovarian cancer cells) were used as target cells, and CAR-X4-NK cells and CAR-X5-NK cells were used as effector cells. The two cell types were co-cultured with SK-OV3 cells at an effector-target ratio of 5:1, and the killing effect of CAR-X4-NK cells and CAR-X5-NK cells on SK-OV3 cells was monitored.

[0082] The results are as follows Figure 6 and Figure 7 As shown, comparative analysis revealed that, with a consistent initial tumor cell count, CAR-X4-NK cells only experienced a gradual decrease in tumor cell count below the initial number after approximately 16 hours of co-culture, while CAR-X5-NK cells required approximately 24 hours of co-culture. Furthermore, CAR-X4-NK cells only began to show a tumor cell count below the number of normally growing tumor cells after approximately 12 hours of co-culture, while CAR-X5-NK cells required approximately 14 hours of co-culture. These results indicate that CAR-X4-NK cells exert their cytotoxic effect earlier than CAR-X5-NK cells.

[0083] Furthermore, when CAR-X4-NK cells and CAR-X5-NK cells were co-cultured with tumor cells for 30 h, the number of tumor cells co-cultured with CAR-X4-NK cells was less. Overall, the killing efficiency of CAR-X4-NK cells against tumor cells was significantly higher than that of CAR-X5-NK cells, indicating that CAR-X4-NK cells have a stronger killing ability. This suggests that CAR cells modified with the "discontinuous truncated functional region" expressing GM-CSF have a better killing effect on tumor cells than CAR cells modified with the "continuous truncated functional region" expressing GM-CSF.

[0084] In the CAR-X5-NK cells involved in this embodiment, the nucleotide sequence of the 5th truncated region of GM-CSF represented by X5 is as follows.

[0085] Table 2

[0086]

[0087] Example 6

[0088] This embodiment further examines the cytokine secretion capacity of CAR-NK cells from Example 5.

[0089] The supernatants of CAR-X4-NK cells and CAR-X5-NK cells were collected after co-culturing with tumor cells for 24 hours, and the IFN-γ and TNF-α in the supernatants were analyzed. The levels of IFN-γ and TNF-α in the supernatant were significantly increased after co-culturing CAR-NK cells with target cells. Figure 8 and Figure 9 The specific results are shown in Table 2.

[0090] Table 3. Results of cytokine secretion by CAR-NK cells.

[0091]

[0092] The results showed that when the effector-target cell ratio was 5:1, the TNF-α and IFN-γ secreted by CAR-X4-NK cells expressing the GM-CSF functional region (discontinuously truncated (X4)) and CAR-X5-NK cells expressing the GM-CSF functional region (continuously truncated (X5)) were significantly increased compared with conventional CAR.

[0093] Furthermore, CAR-X4-NK cells secreted 2.4 times more TNF-α than CAR-X5-NK cells, while the secretion of IFN-γ was comparable. Overall, the CAR-X4-NK cells of this invention exhibit better cytokine secretion capabilities. This suggests that, under the premise of expressing the same number of functional regions, the modification method expressing discontinuous truncated functional regions significantly enhances the CAR effect compared to the modification method expressing continuous truncated functional regions.

[0094] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0095] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A CAR-NK cell expressing a discontinuously truncated functional region of GM-CSF, characterized in that, The CAR-NK cells express a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises a discontinuous truncated functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the discontinuous truncated functional region fragment of the GM-CSF protein includes an A helix fragment, a C helix fragment, and an exon 2 fragment; the discontinuous truncated functional region fragment of the GM-CSF protein is linked to the chimeric antigen receptor via a P2A sequence; 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 signaling region, and a human CD3ζ signaling domain; the nucleotide sequence encoding the discontinuous truncated functional region fragment of the GM-CSF protein is shown in SEQ ID NO.1; the nucleotide sequence encoding the chimeric antigen receptor expressed by the CAR-NK cells is shown in SEQ ID NO.

3.

2. The CAR-NK cells as described in claim 1, characterized in that, The nucleotide encoding the novel chimeric antigen receptor structure is also linked to an EF1-α promoter sequence.

3. The use of the CAR-NK cells according to claim 1 or 2 in the preparation of a medicament for treating solid tumors.

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