Immune engineering cell for expressing exon 2 functional area 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 an exon 2 functional area of GM-CSF and application. The invention firstly provides a CAR-NK cell which expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises an exon 2 functional domain fragment of a GM-CSF protein and a chimeric antigen receptor. The invention also provides a CAR-T cell of the exon 2 functional domain fragment which also expresses the GM-CSF. The invention provides a novel chimeric antigen receptor structure modified by a fragment only containing a unique functional region in the full-length GM-CSF, which not only retains the function equivalent to that of the traditional CAR and CAR-GM, but also obtains an improved function.

Description

[0001] Priority application This application claims the priority of the Chinese invention patent application [CN2023115953752] "[A CAR-T comprising the third truncated region of GM-CSF and its preparation method and application]" filed on November 24, 2023, and the priority of [CN2023115953911] "[A CAR-T comprising the fourth truncated 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 specifically relates to an immune engineering cell expressing the exon 2 functional region of GM-CSF and an application thereof. Background Art

[0003] 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.

[0004] 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. 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.

[0005] 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.

[0006] In summary, it is necessary to make improvements to the existing technology. Summary of the invention

[0007] The purpose of the present invention is to provide an immunoengineered cell expressing the exon 2 functional region of GM-CSF and its application. The specific technical scheme is as follows.

[0008] A CAR-NK cell expressing the exon 2 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 fragment of the exon 2 functional region of the GM-CSF protein and a chimeric antigen receptor; the exon 2 functional region fragment 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.

[0009] Preferably, the nucleotide sequence of the exon 2 functional region fragment is shown in SEQ ID NO.1.

[0010] Furthermore, the novel chimeric antigen receptor structure is also connected to an EF1-α promoter sequence.

[0011] Preferably, the nucleotide sequence of the chimeric antigen receptor expressed by the CAR-NK cell is as shown in SEQ ID NO.3.

[0012] A CAR-T cell expressing the exon 2 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 fragment of the exon 2 functional region of the GM-CSF protein and a chimeric antigen receptor; the exon 2 functional region fragment 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 CD3ζ signal domain.

[0013] Preferably, the nucleotide sequence of the exon 2 functional region fragment is shown in SEQ ID NO.1.

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

[0015] Preferably, the nucleotide sequence of the chimeric antigen receptor expressed by the CAR-T cell is as shown in SEQ ID NO.3.

[0016] The present invention can also provide the use of the above-mentioned CAR-NK cells in the preparation of drugs for treating solid tumors.

[0017] 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, CAR-NK cells have a broad spectrum of highly effective tumoricidal activity. (3) Allogeneic re-infusion therapy can be achieved.

[0018] The use of the above-mentioned CAR-T cells in the preparation of drugs for treating solid tumors.

[0019] Furthermore, the tumor includes hematological tumor, liver cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, ovarian cancer, glioma, pancreatic cancer, sarcoma or glioblastoma.

[0020] The above-mentioned CAR-T cells or CAR-NK cells can enhance the killing effect on tumor cells by expressing exon 2 of GM-CSF protein.

[0021] The use of the above-mentioned CAR-NK cells in the preparation of TNF-α promoters.

[0022] Preferably, the CAR-NK cells can act as a TNF-α promoter to secrete more TNF-α.

[0023] The use of the above-mentioned CAR-NK cells in the preparation of IFN-γ promoters.

[0024] Preferably, the CAR-NK cells can act as an IFN-γ promoter to secrete more IFN-γ.

[0025] The antigen binding domain in the CAR provided by the present invention can target CD19, BCMA, HER2, Claudin18.2, Mesothelin, GPC3 or GD2, etc.

[0026] 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 the exon 2 functional region of GM-CSF; the nucleic acid encoding the chimeric antigen receptor and the nucleic acid encoding the exon 2 functional region of GM-CSF are connected through a sequence encoding 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; the lentivirus includes pWPXLD, psPAX2 and pMD2.G.

[0027] 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.

[0028] Beneficial technical effects: On the one hand, the present invention constructs an immune engineering cell expressing exon 2, the only functional region of GM-CSF, which expresses a novel chimeric antigen receptor structure. Compared with the full-length protein of GM-CSF, the novel chimeric antigen receptor structure of the present invention only contains one of the functional regions of the full-length protein of GM-CSF, which reduces the sequence length of the overexpressed gene, but does not affect the expression and function of CAR. On the contrary, since the truncated functional region does not contain a signal peptide, it can function intracellularly, resulting in an increase in the proliferation ability of CAR-T cells. The proliferation ability of the CAR-X3-T cells of the present invention is better than that of conventional CAR-T cells and CAR-GM-T cells. Obviously, the CAR-X3-T cells of the present invention have higher yields and lower production costs when used for industrial production.

[0029] On the other hand, the experiment of the present invention confirmed that under the premise of containing the same GM-CSF truncated functional region, the effect of reducing the number of functional regions on the results (that is, the effect of only containing the only functional region and containing other functional regions other than the only functional region on the results). The results confirmed that the CAR containing only the only functional region exon 2 obtained by the present invention, as a modification containing only the minimum functional region fragment in the full-length GM-CSF, not only retains the functions equivalent to those of traditional CAR and CAR-GM, but also achieves improved functions. Specifically, the CAR-X3-T cells prepared by the present invention have a significantly stronger killing ability against tumors than conventional CAR-T as a whole, and are equivalent to the effect of GM-CSF full-length CAR-T, and are better than CAR-X4-T cells containing 3 functional region fragments; more cytokine secretion can be detected in the supernatant of co-culture of CAR-X3-NK cells prepared by the present invention and tumor cells. Therefore, the CAR-X3-NK cells or CAR-X3-T cells of the present invention have the potential to become adjuvant therapy products and play a role in the preparation of anti-tumor therapeutic drugs.

[0030] 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

[0031] 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.

[0032] 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-X3-T cells in one of the embodiments of the present invention; Figure 4 This is a graph showing the proliferation results of CAR-X3-T cells and control cells (CAR-X4-T) in one of the embodiments of the present invention; Figure 5 It showed that CAR-X3-T cells and control cells (CAR-X4-T) had higher killing efficiency against tumor cells than conventional CAR-T cells; Figure 6 It showed that CAR-X3-NK cells secreted 59.6 times more TNF-α and 1.7 times more IFN-γ than conventional CAR-NK cells; Figure 7 The killing curve of tumor cells by CAR-X3-NK cells is shown. DETAILED DESCRIPTION

[0033] 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.

[0034] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0035] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0036] 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.

[0037] 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.

[0038] The "novel chimeric antigen receptor structure" described in the present invention refers to a chimeric antigen receptor (CAR) and a functional region fragment (X3) 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.

[0039] 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.

[0040] Based on the above transformation methods, the full-length GM-CSF protein was prepared into 5 short peptides containing different truncated fragments, including: The first truncation region of GM-CSF mainly includes the A helix region of GM-CSF, which is represented by X1.

[0041] The second truncation region of GM-CSF mainly includes exon 1 and Chelix region of GM-CSF, which is represented by X2.

[0042] The third truncation region of GM-CSF mainly includes the exon 2 region of GM-CSF, which is represented by X3.

[0043] The fourth truncation region of GM-CSF mainly includes the A helix, Chelix and exon 2 regions of GM-CSF, represented by X4.

[0044] The fifth truncation region of GM-CSF mainly includes the A helix, exon 1 and Chelix regions of GM-CSF, which is represented by X5.

[0045] 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.

[0046] Another invention is to explore the minimum truncated unit that can maintain the function of CAR and has certain functional improvements under the premise of truncating the full-length GM-CSF protein.

[0047] The sequence information involved in the present invention is shown in Table 1.

[0048] Table 1 Example 1 Construction of novel CAR expression vector and viral packaging 1.1 Construction of lentiviral expression vector for novel CAR vector The lentiviral expression vector pWPXLD-CAR-P2A-X3 expressing the novel CAR was constructed by molecular cloning. The gene fragment of the expression vector includes CD8 signal peptide, SCFV, hinge region (hinge), human CD8 transmembrane region (TM), human 4-1BB costimulatory signal region, human CD3 ζ signal domain, P2A, fragment (X3), such as Figure 1 Among them, X3 mainly contains the exon 2 region of GM-CSF and does not contain a signal peptide, which means that it can enhance the function of CAR in the cell.

[0049] 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.

[0050] Example 2 CAR positive rate detection The lentivirus in Example 1 was used to transduce T cells to obtain novel CAR-T cells. The target protein expressing the His tag was used as the primary antibody, and the His antibody coupled to the fluorescent APC was used as the secondary antibody to detect the CAR positive rate. Conventional CAR is a traditional second-generation CAR-T, CAR-GM is a CAR containing the full-length GM, and CAR-X3 is a CAR containing the fragment X3. The results are shown in Figure 1. Figure 3 The results showed that the positive rate of CAR-X3-T reached 58%, which was not much different from that of traditional CAR-T cells and CAR-GM-T cells, indicating that CAR containing only one functional region, exon 2, still retained a transfection rate comparable to that of traditional CAR and CAR-GM.

[0051] Example 3 This example detects the proliferation ability of the CAR-X3-T cells synthesized in Example 2.

[0052] The same number of T cells were transduced with the lentivirus in Example 1 to obtain novel CAR-X3-T cells. After 3 days, the number of CAR-X3-T cells was detected using a cell counter and the expansion multiple was calculated. The results are as follows: Figure 4 As shown. The results showed that the proliferation fold of traditional CAR-T cells was the lowest; the proliferation fold of CAR-GM-T cells was higher than that of traditional CAR-T cells; and the proliferation fold of CAR-X3-T cells was the highest. This indicates that CAR containing only the unique functional region exon 2 not only retains the same transfection rate as traditional CAR and CAR-GM, but also the CAR-X3-T prepared by it has the highest proliferation fold in the same period of time.

[0053] In order to further explore the effect of the truncated functional region of GM-CSF on cell proliferation, this example also provides the following control experiment.

[0054] The same number of T cells were transduced using a similar method as in Example 1 to construct CAR-X4-T (control cells), where the CAR structures of CAR-X4-T and CAR-X3-T were the same. The X4 fragment contained three functional regions of the A helix fragment, the Chelix fragment, and the exon 2 fragment of GM-CSF, as shown in the following examples.

[0055] Table 2 After 3 days, the proliferation of CAR-X3-T cells and CAR-X4-T cells was detected using a cell counter and the expansion times were calculated. Figure 4. The results showed that the proliferation ability of CAR-XT cells prepared by truncating the full-length GM-CSF and retaining the necessary functional regions was stronger than that of CAR-T cells prepared by full-length GM-CSF. However, based on the functional region of X4, the functional region was further deleted to obtain X3, which only contained one functional region fragment. Although the effect on CAR-T cell proliferation was smaller than that of CAR-X4-T, it was generally stronger than that of traditional CAR-T and CAR-GM-T. This shows that the CAR proposed in the present invention, which only contains the only functional region exon 2, as a modification containing only the minimum functional region fragment in the full-length GM-CSF, not only retains the functions equivalent to those of traditional CAR and CAR-GM, but also achieves improved functions.

[0056] In the CAR-X4-T cells involved in this embodiment, the nucleotide sequence of X4 is as follows.

[0057] Table 3 Example 4 This example detects the verification that the CAR-X3-T cells synthesized in Example 2 kill tumor cells.

[0058] CAR target-positive SK-OV3 cells (human ovarian cancer cells) were used as target cells, and CAR-X3-T cells were used as effector cells. The two cell types were co-cultured at an effector-target ratio of 5:1, and the killing of SK-OV3 cells by CAR-X3-T cells was monitored.

[0059] like Figure 5 As shown in the figure, it can be seen that from about 6 hours onwards, the killing ability of CAR-X3-T cells and CAR-GM-T cells on tumor cells is higher than that of conventional CAR-T cells. After about 10 hours, this killing advantage is more significant. The killing effect of the killing curve for 40 hours was analyzed, and the results showed that the killing effect of CAR-X3-T cells on tumor cells was equivalent to that of CAR-GM-T cells.

[0060] To further explore the effect of GM-CSF truncated functional region on cell proliferation, this example also used CAR-X4-T cells as control cells for control experiments. The results showed that ( Figure 5 ), there was no significant difference between CAR-X3-T cells and CAR-X4-T cells in killing tumor cells.

[0061] Example 5 The lentivirus in Example 1 was further used to transduce NK cells to obtain novel CAR-X3-NK cells. The supernatants of CAR-X3-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-NK cells were co-cultured with target cells, IFN-γ and TNF-α in the supernatants increased significantly ( Figure 6 ), the specific results are shown in the following table.

[0062] Table 4 CAR-X3-NK cell detection results.

[0063] The results showed that TNF-α secreted by CAR-X3-NK cells was 59.6 times that of conventional CAR-NK cells; CAR-NK cells in the control group hardly expressed TNF-α. IFN-γ was 1.7 times that of conventional CAR-NK cells. This proved that X3, as a modified selection of the minimal functional region fragment of GM-CSF, played the role of an immunomodulatory factor, significantly increasing the expression of IFN-γ and TNF-α of CAR-NK cells compared with the control group.

[0064] Furthermore, based on the novel CAR-X3-NK cells obtained above, SK-OV3 cells with positive CAR target were used as target cells, and CAR-X3-NK cells were used as effector cells. The two cells were co-cultured at an effector-target ratio of 5:1 to monitor the killing of SK-OV3 cells by CAR-X3-NK cells.

[0065] like Figure 7 As shown, the killing efficacy of the killing curve was analyzed for 30 hours, and the results showed that the killing ability of CAR-X3-NK cells against tumor cells was slightly weaker than that of conventional CAR-NK cells.

[0066] The above results show that the CAR-NK cells obtained by the present invention, which contain the only functional region of GM-CSF protein exon 2, have a stronger cytokine secretion ability than conventional CAR-NK cells, but their killing effect on tumor cells is not necessarily better than that of conventional CAR-NK cells. Therefore, the CAR-NK cells of the present invention have the potential to prepare IFN-γ promoters and TNF-α promoters.

[0067] 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.

[0068] 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 the exon 2 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 an exon 2 functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the exon 2 functional region fragment 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 exon 2 functional region fragment 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 the exon 2 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 an exon 2 functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the exon 2 functional region fragment 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 1, wherein The nucleotide sequence of the exon 2 functional region fragment is shown in SEQ ID NO.

1.

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

8. The CAR-T cell according to claim 1, 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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