Immune engineering cell for expressing discontinuous 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 discontinuous truncated functional region of GM-CSF and application. The invention firstly provides a CAR-NK cell for expressing a discontinuous truncated functional region of GM-CSF, and the CAR-NK cell expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises a discontinuous truncated functional domain fragment of a GM-CSF protein and a chimeric antigen receptor; the non-continuous truncated functional domain fragment of the GM-CSF protein comprises an A helix fragment, a C helix fragment and an exon 2 fragment. The invention also provides a CAR-T cell for expressing the discontinuous truncated functional region of the GM-CSF as well.

Description

[0001] Priority application This application claims priority to the Chinese invention patent application [CN2023115953911] filed on November 24, 2023, "A CAR-T comprising the 4th truncation region of GM-CSF, and preparation method and application", and the Chinese invention patent application [CN2023115954083] filed on November 24, 2023, "A CAR-T comprising the 5th truncation region of GM-CSF, and 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 discontinuous 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 certain target antigen epitope, thereby enhancing the lymphocyte recognition of antigen signals and activation functions. 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. Based on the first-generation CAR-T, the second-generation CAR-T was developed. 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 immunomodulatory factors 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). The intracellular signal activation domains of CAR-T cells are mainly 41BB, CD28 and CD3 ζ, so the traditional CAR molecule design that works on CAR-T may not necessarily play a good activation role in NK cells.

[0008] In summary, it is necessary to make improvements to 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 a non-continuous truncated functional region of GM-CSF, and a preparation method and application thereof. The specific technical scheme is as follows.

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

[0011] A CAR-NK cell expressing a discontinuous 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 discontinuous truncated functional region fragment of GM-CSF protein and a chimeric antigen receptor; the discontinuous truncated functional region fragment of GM-CSF protein comprises an A helix fragment, a Chelix fragment and an exon 2 fragment; the discontinuous truncated functional region fragment of 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.

[0012] Furthermore, the nucleotide sequence of the non-continuous truncated functional region fragment 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 a non-continuous truncated functional region of GM-CSF.

[0016] A CAR-T cell expressing a discontinuous 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 discontinuous truncated functional region fragment of GM-CSF protein and a chimeric antigen receptor; the discontinuous truncated functional region fragment of GM-CSF protein comprises an A helix fragment, a Chelix fragment and an exon 2 fragment; the discontinuous truncated functional region fragment of 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.

[0017] Furthermore, the nucleotide sequence of the non-continuous truncated functional region fragment 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 use of the above CAR-NK cells in the preparation of drugs for treating solid tumors. The use of the above CAR-T cells in the preparation of drugs for treating solid tumors.

[0021] 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 re-infusion therapy can be achieved.

[0022] The above-mentioned CAR-NK cells or CAR-T cells can enhance the killing effect on tumor cells by expressing non-continuous truncated functional regions of GM-CSF.

[0023] The antigen binding domain in the CAR provided by the present invention can target CD19, BCMA, HER2, Claudin18.2, Mesothelin, GPC3, GD2, etc.

[0024] Furthermore, the tumor includes hematological tumor, liver cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, ovarian cancer, glioma, pancreatic cancer, sarcoma or glioblastoma.

[0025] The present invention may also include a method for preparing the above-mentioned CAR or the above-mentioned CAR-T or the above-mentioned CAR-NK.

[0026] Beneficial technical effects: On the one hand, the present invention constructs an immune engineering cell expressing a non-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 non-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 non-continuous truncated functional region does not contain a signal peptide, it can function intracellularly and improve the proliferation ability of CAR-T cells. The proliferation ability of the CAR-X4-T cells of the present invention is significantly better than that of conventional CAR-T cells and CAR-GM-T cells. Obviously, the CAR-X4-T cells of the present invention have higher yields and lower production costs when used for industrial production, and have greater industrial promotion value.

[0027] On the other hand, the experiment of the present invention confirms 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. The present invention confirms that the CAR-NK cells (CAR-X4-NK) modified with non-continuous truncated functional regions expressing GM-CSF are more efficient in killing tumor cells than the CAR-NK cells (CAR-X5-NK) modified with continuous truncated functional regions expressing GM-CSF, and their cytokine secretion ability is stronger as a whole, and the enhancement of CAR effect is more significant. Specifically, the CAR-X4-NK cells of the present invention kill tumor cells 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. In addition, 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 better cytokine secretion ability than CAR-X5-NK cells. Therefore, the CAR-X4-NK cells of the present invention can be used as a cell therapy auxiliary product and play a greater role in the preparation of anti-tumor therapeutic drugs.

[0028] Finally, due to the differences between NK cell modification and T cell modification, the CAR structure expressed and functional on CAR-T cells does not necessarily have the same effect on CAR-NK cells, which requires experimental verification. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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.

[0030] 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 3 This is a result diagram of the CAR positivity detection of CAR-X4-T cells constructed in one of the embodiments of the present invention; Figure 4 This is a result diagram of the proliferation of CAR-X4-T cells constructed in one of the embodiments of the present invention; Figure 5 This is a killing curve of tumor cells by CAR-X4-T cells constructed in one of the embodiments of the present invention; Figure 6 This is a killing curve of tumor cells by CAR-X4-NK cells constructed in one embodiment of the present invention; Figure 7 This is a killing curve diagram of tumor cells by CAR-X5-NK cells of the control group constructed in one of the embodiments of the present invention; Figure 8 Figure 2 shows the TNF-α detection results of CAR-X4-NK cells and control group CAR-X5-NK cells after co-culture with tumor cells when the effector-target cell ratio was 5:1 (A: TNF-α secretion results of CAR-X4-NK cells, B: TNF-α secretion results of CAR-X5-NK cells); Fig. 9 Figure 2 is the IFN-γ detection results after co-culture of CAR-X4-NK cells and control group CAR-X5-NK cells with tumor cells when the effector-target cell ratio was 5:1 (A: IFN-γ secretion results of CAR-X4-NK cells; B: IFN-γ secretion results of CAR-X5-NK cells). DETAILED DESCRIPTION

[0031] 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.

[0032] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0033] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0034] 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.

[0035] 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.

[0036] Definition of noun: The "novel chimeric antigen receptor structure" described in the present invention refers to a non-continuous truncated functional region fragment (X4) 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.

[0037] 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 peptid fragments, A helix 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.

[0038] 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 of GM-CSF, indicated by X1.

[0039] GM-CSF second truncation region: includes the exon 1 fragment region and the Chelix fragment region of GM-CSF, represented by X2.

[0040] GM-CSF third truncation region: contains only the exon 2 fragment of GM-CSF, represented by X3.

[0041] GM-CSF 4th truncation region: includes the A helix, Chelix and exon 2 fragments of GM-CSF, represented by X4.

[0042] GM-CSF 5th truncation region: includes the A helix, exon 1 and Chelix fragments of GM-CSF, represented by X5.

[0043] 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.

[0044] Another invention is to explore the effects of containing continuous truncated functional regions and containing non-continuous truncated functional regions on CAR function under the premise of containing the same number of functional regions.

[0045] The "continuously truncated functional region" mentioned in the present invention refers to the 5th truncated region, represented by X5; the "non-continuously truncated functional region" mentioned in the present invention refers to the 4th truncated region, represented by X4.

[0046] The sequence information involved in the present invention is shown in Table 1.

[0047] Table 1 Example 1 Construction and viral packaging of a novel CAR expression vector.

[0048] 1.1 Construction of lentiviral expression vector of novel CAR vector.

[0049] The lentiviral expression vector pWPXLD-CAR-P2A-X4 expressing the novel CAR was constructed by molecular cloning. The gene fragment of the expression vector includes CD8 signal peptide, single-chain antibody (SCFV), hinge region (hinge), human CD8 transmembrane region (TM), human 4-1BB costimulatory signal region, human CD3 ζ signal domain, P2A, fragment (X4), such as Figure 1 shown.

[0050] 1.2 Packaging of novel CAR lentivirus 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.

[0051] Example 2 CAR positivity rate detection.

[0052] The lentivirus in Example 1 was used to transduce T cells to obtain novel CAR-X4-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. Conventional CAR is a traditional second-generation CAR-T, CAR-GM is a CAR containing the full-length GM, and CAR-X4 is a CAR containing the fragment X4. The results are shown in Figure 3 As shown, the positive rate of CAR in CAR-X4-T cells was 56.75%, which was comparable to that of conventional CAR and CAR-GM, indicating that only part of the GM-CSF functional region was contained, and the functional region was a non-continuous truncated region, which would not affect the expression of CAR.

[0053] Example 3 This example detects the proliferation ability of the CAR-X4-T cells synthesized in Example 1.

[0054] The same number of T cells were transduced using the lentivirus in Example 1 to obtain novel CAR-X4-T cells. After 3 days, the number of CAR-X4-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-X4-T cells is significantly better than that of conventional CAR-T cells and CAR-GM-T cells, proving that reducing the length of GM-CSF improves cell proliferation ability.

[0055] Example 4 This example tests the verification that the CAR-X4-T cells synthesized in Example 1 kill tumor cells.

[0056] CAR target-positive SK-OV3 cells (human ovarian cancer cells) were used as target cells, and the CAR-X4-T cells obtained in Example 4 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-X4-T cells was monitored.

[0057] The results are as follows Figure 5 As shown, it can be seen that from about 5 hours onwards, the killing ability of CAR-X4-T cells on tumor cells is significantly higher than that of conventional CAR. Analysis of the killing efficacy of the killing curve for 40 hours showed that the killing efficacy of CAR-X4-T cells on tumor cells is comparable to that of CAR-GM-T cells and higher than that of conventional CAR.

[0058] The above experimental results show that reducing the length of GM-CSF can enhance the proliferation ability of CAR-T cells without affecting the killing effect on tumors, which is equivalent to CAR-T prepared with full-length GM-CSF.

[0059] Example 5 This example provides verification of CAR-X4-NK cells killing tumor cells.

[0060] 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 structures of CAR-X4-NK and CAR-X5-NK were consistent.

[0061] CAR target-positive SK-OV3 cells (human ovarian cancer cells) were used as target cells, CAR-X4-NK cells and CAR-X5-NK cells were used as effector cells, respectively. The two types of cells were co-cultured with SK-OV3 cells at an effector-target ratio of 5:1, and the killing of SK-OV3 cells by CAR-X4-NK cells and CAR-X5-NK cells was monitored.

[0062] The results are as follows Figure 6 and Figure 7 As shown in the figure, through comparative analysis, it was found that when the initial number of tumor cells was the same, the number of tumor cells of CAR-X4-NK cells gradually decreased to below the initial number of cells only after about 16 hours of co-culture, while CAR-X5-NK cells needed to be co-cultured for about 24 hours. In addition, the number of tumor cells of CAR-X4-NK cells gradually began to be lower than the number of normally growing tumor cells only after about 12 hours of co-culture, while CAR-X5-NK cells needed to be co-cultured for about 14 hours. The above results show that CAR-X4-NK cells have a killing effect earlier than CAR-X5-NK cells.

[0063] In addition, when CAR-X4-NK cells and CAR-X5-NK cells were co-cultured with tumor cells for 30 h, respectively, the number of tumor cells co-cultured with CAR-X4-NK cells was smaller. 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 stronger killing ability, suggesting that the CAR modified with "non-continuous truncated functional region" expressing GM-CSF has a better killing effect on tumor cells than the CAR modified with "continuous truncated functional region" expressing GM-CSF.

[0064] In the CAR-X5-NK cells involved in this embodiment, the nucleotide sequence of the 5th truncation region of GM-CSF represented by X5 is as follows.

[0065] Table 2 Example 6 This example further detects the cytokine secretion ability of the CAR-NK cells of Example 5.

[0066] The supernatants of CAR-X4-NK cells and CAR-X5-NK cells were collected after co-culture with tumor cells for 24 hours, and the IFN-γ and TNF-α in the supernatants were analyzed. The IFN-γ and TNF-α in the supernatants increased significantly after CAR-NK cells were co-cultured with target cells ( Figure 8 and Fig. 9 ), the specific results are shown in Table 2.

[0067] Table 3 Detection results of cytokines secreted by CAR-NK cells.

[0068] The results showed that when the effector-target cell ratio was 5:1, CAR-X4-NK cells expressing discontinuous truncation (X4) of the GM-CSF functional region and CAR-X5-NK cells expressing continuous truncation (X5) of the GM-CSF functional region respectively secreted significantly more TNF-α and IFN-γ than conventional CAR.

[0069] Furthermore, the TNF-α secreted by CAR-X4-NK cells is 2.4 times that of CAR-X5-NK cells, and the secretion of IFN-γ is comparable. Overall, the CAR-X4-NK cells of the present invention have better cytokine secretion ability. It is suggested that under the premise of expressing the same number of functional regions, the transformation method of expressing non-continuous truncated functional regions is more significant than the transformation method of expressing continuous truncated functional regions in enhancing the CAR effect.

[0070] 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.

[0071] 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 non-continuous truncated functional region of GM-CSF, characterized in that: The CAR-NK cell 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 Chelix fragment and an exon 2 fragment; the non-continuous truncated functional region fragment of GM-CSF protein is connected to the chimeric antigen receptor through 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 non-continuous 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, characterized in that 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 a non-continuous truncated functional region of GM-CSF, characterized in that: The CAR-T cell 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 Chelix fragment and an exon 2 fragment; the non-continuous truncated functional region fragment of 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, characterized in that The nucleotide sequence of the non-continuous 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, characterized in that 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 drugs for treating solid tumors.

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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