Immunologically engineered cells expressing truncated functional domain fragments of gm-csf and uses thereof

Abstract

The application belongs to the technical field of biology and particularly relates to immune engineering cells expressing GM-CSF truncated functional region fragments and application. The application first provides a CAR-NK cell expressing a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises a GM-CSF protein truncated functional region fragment and a chimeric antigen receptor; the GM-CSF protein truncated functional region fragment comprises an exon 1 functional region fragment and a C helix functional region fragment. The application also provides a CAR-T cell also expressing a GM-CSF truncated functional region fragment. The novel immune engineering cell provided by the application not only retains functions equivalent to conventional CAR and full-length CAR-GM CAR, but also achieves improved functions.

Description

[0001] Priority Application

[0002] This application claims priority to Chinese Invention Patent Application

CN2023115817245

[0003] The present application belongs to the field of biotechnology, and specifically relates to an immune engineering cell expressing a GM-CSF truncated functional region fragment and application thereof. BACKGROUND

[0004] Chimeric antigen receptor (CAR) is an artificial receptor molecule manufactured by genetic engineering technology, which can endow immune effector cells (such as T cells, NK cells) with specificity for a certain target antigen epitope, thereby enhancing the function of lymphocytes in recognizing antigen signals and activation. The first generation of CAR has only CD3 ξ (containing 3 ITAM) and FcRγ (containing 2 ITAM) signaling domains in the intracellular, which can only provide the first signal of T cell activation. Early clinical trials of the first generation of CAR showed that the first generation of CAR-T could not effectively maintain the proliferation and activation of T cells, and satisfactory results were not achieved, indicating that the first generation of CAR-T cells may lack sufficient activation signals to maintain T cell proliferation and effective anti-tumor effect. On the basis of the first generation of CAR-T, the second generation of CAR-T was developed. The second generation of CAR-T cells showed enhanced in vivo expansion and persistence ability, and its effect has been confirmed in clinical trials.

[0005] Unfortunately, compared with the good effect on hematological tumors, most of the reported effects of CAR-T cell therapy on solid tumors have always been difficult to effectively break through at some key stages. Therefore, there is still a need for technical innovation in this field.

[0006] NK cells are an important member of the innate immune system, mainly exist in blood and lymphoid organs, do not need to be pre-sensitized, and have no major histocompatibility complex (MHC) restriction, can quickly and directly kill target cells, and have a broad spectrum of anti-tumor effect. NK cells kill target cells in many ways, once the balance between inhibitory signals and activating 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 expression of chimeric antigen receptor (CAR) on the surface of NK cells can significantly improve the anti-cancer effect of immune cells. At present, the scientific community believes that compared with CAR-T cells, CAR-NK cells have some significant advantages. However, the preparation of CAR-NK cells with tumor killing efficiency has some difficulties, mainly in that NK cells have natural antiviral function, and it is difficult to genetically modify them.

[0007] The patent with publication number CN114934071A and the invention name of "CAR vector expressing immunomodulatory factor and application thereof" 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 activating receptors include natural cytotoxicity receptors (NCR), such as NKG2D, CD16 (FcgRIIIa), FasL and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and costimulatory receptors, such as LFA-1, CD244 (2B4) and CD137 (41BB). The intracellular signal activation domain of CAR-T cell is mainly 41BB, CD28 and CD3zeta, 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 prior art. SUMMARY

[0009] The purpose of the present application is to provide an immune engineering cell expressing a GM-CSF truncated functional region fragment and its application, and the specific technical solutions are as follows.

[0010] In one aspect, the present application provides a novel CAR-NK cell.

[0011] A CAR-NK cell expressing a truncated functional domain fragment of GM-CSF, the CAR-NK cell expressing a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprising a truncated functional domain fragment of GM-CSF protein and a chimeric antigen receptor; the truncated functional domain fragment of GM-CSF protein comprising an exon 1 functional domain fragment and a C helix functional domain fragment; the truncated functional domain fragment of GM-CSF protein being connected with the chimeric antigen receptor through a P2A sequence; the chimeric antigen receptor comprising 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 zeta signal domain.

[0012] As a preferred, the nucleotide sequence of the truncated functional domain fragment of GM-CSF protein is shown as SEQ ID NO. 1.

[0013] Further, the novel chimeric antigen receptor structure is also connected with an EF1-alpha promoter sequence.

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

[0015] Another aspect of the present application provides a novel CAR-T cell.

[0016] A CAR-T cell expressing a truncated functional domain fragment of GM-CSF, the CAR-T cell expressing a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprising a truncated functional domain fragment of GM-CSF protein and a chimeric antigen receptor; the truncated functional domain fragment of GM-CSF protein comprising an exon 1 functional domain fragment and a C helix functional domain fragment; the truncated functional domain fragment of GM-CSF protein being connected with the chimeric antigen receptor through a P2A sequence; the chimeric antigen receptor comprising 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 zeta signal domain.

[0017] As a preferred, the nucleotide sequence of the truncated functional domain fragment of GM-CSF protein is shown as SEQ ID NO. 1.

[0018] Further, the novel chimeric antigen receptor structure is also connected with an EF1-alpha promoter sequence.

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

[0020] The application also provides the application of the new CAR-NK cell and the new CAR-T cell.

[0021] The application of the CAR-NK cell in the preparation of an immune enhancer for assisting tumor treatment.

[0022] The application proves that the content of immune regulatory factors INF-γ and TNF-α contained in the supernatant of the co-culture of the CAR-NK cell expressing the truncated functional region fragment of GM-CSF and the tumor is significantly higher than that of the ordinary CAR-NK cell, so the CAR-NK cell expressing the truncated functional region fragment of GM-CSF has great potential in the preparation of an immune enhancer for assisting tumor treatment.

[0023] Further, the application also provides the application of the CAR-NK cell in the preparation of a drug for treating a solid tumor.

[0024] Compared with the CAR-T cell, the CAR-NK cell has some significant advantages, including: (1) less cytokine release syndrome and neurotoxicity; (2) the CAR-NK cell can kill cancer cells through two pathways of CAR dependence and CAR independence. In addition, the NK cell can kill tumor cells through CD16-mediated ADCC. Therefore, the CAR-NK cell has broad-spectrum and high-efficiency tumor killing activity; and (3) the allogeneic reinfusion therapy can be realized.

[0025] The application of the CAR-T cell in the preparation of a drug for treating a solid tumor.

[0026] The CAR-NK cell or the CAR-T cell can enhance the killing effect on tumor cells by expressing the truncated functional region fragment (exon 1+ Chelix) of the GM-CSF protein.

[0027] The antigen binding domain in the CAR provided by the application can be targeted to CD19, BCMA, HER2, Claudin18.2, Mesothelin, GPC3 or GD2, etc.

[0028] Further, the tumor includes a hematological tumor, a liver cancer, a breast cancer, a lung cancer, an esophageal cancer, a gastric cancer, an ovarian cancer, a glioma, a pancreatic cancer, a sarcoma or a glioblastoma.

[0029] The application can also include a preparation method of the CAR or a preparation method of the CAR-T cell or the CAR-NK cell.

[0030] Beneficial technical effects:

[0031] The application constructs an immune engineering cell expressing a truncated functional region of GM-CSF, and the immune engineering cell expresses a novel chimeric antigen receptor structure. Compared with a CAR containing a full-length GM-CSF protein, the novel chimeric antigen receptor structure of the application only increases a short peptide of about 50 amino acids in the CAR structure, which can significantly improve the in vitro infection efficiency and proliferation capacity of CAR-T, and the anti-tumor cell capacity is flat with that of CAR-T prepared by full-length GM-CSF, but the sequence length of the overexpressed gene is reduced, and thus some potential risks are reduced. Due to the higher proliferation capacity, the CAR-X2-T cells of the application have higher yield and lower production cost when used for industrialized production.

[0032] Specifically, the CAR-X2-T cells prepared by the application have better overall killing ability on tumors than conventional CAR-T; compared with conventional CAR-NK, more cytokine secretion can be detected in the supernatant of the co-culture of CAR-X2-NK cells and tumor cells. Therefore, the CAR-X2-T cells or CAR-X2-NK cells of the application have the potential to become an adjuvant therapy product and play a role in the preparation of anti-tumor therapeutic drugs.

[0033] Finally, due to the difference between NK cell modification and T cell modification, the CAR structure expressed and functioning on CAR-T cells may not have the same effect on CAR-NK cells, and needs to be verified by experiments. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. In all the drawings, similar elements or parts are generally identified by similar reference signs. In the drawings, each element or part is not necessarily drawn according to the actual proportion. Obviously, the drawings described below are some embodiments of the application, and those skilled in the art can obtain other drawings according to these drawings without paying creative labor.

[0035] Figure 1 A schematic diagram of the novel CAR structure of the application;

[0036] Figure 2 A structural schematic diagram of the full-length protein functional region of CM-CSF;

[0037] Figure 3 A result diagram of CAR positive rate detection of CAR-X2-T cells in one embodiment of the application;

[0038] Figure 4 A proliferation result diagram of CAR-X2-T cells in one embodiment of the application;

[0039] Figure 5 For one of the embodiments of the present application, the killing efficiency of CAR-X2-T cells on tumor cells is higher than that of the control group CAR-T cells;

[0040] Figure 6 For one of the embodiments of the present application, the killing ability of CAR-X2-NK cells on tumor cells is higher than that of conventional CAR-T cells as a whole;

[0041] Figure 7 For one of the embodiments of the present application, the CAR-X2-NK cells secrete more TNF-α and IFN-γ than conventional CAR-NK cells. DETAILED DESCRIPTION

[0042] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0043] In this document, "and / or" includes any and all combinations of one or more of the associated items.

[0044] In this document, "a plurality of" means two or more, that is, it includes two, three, four, five, and so on.

[0045] 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, even more typically + / - 0.5% of the stated value.

[0046] In this specification, certain embodiments can be disclosed in one format in terms of a range. It is to be understood that such a "range" description is for convenience and brevity and should not be construed as limiting the scope of the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within the 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, and individual numbers within the range, such as 1, 2, 3, 4, 5, and 6. The above rule applies regardless of the breadth of the range.

[0047] Name interpretation:

[0048] The "new chimeric antigen receptor structure" described in the present application refers to one of the truncated functional region fragments (X2) of the chimeric antigen receptor (CAR) and the GM-CSF protein connected by 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 costimulatory signal region and a human CD3 zeta signal domain.

[0049] The "GM-CSF truncated functional region" described in the present application refers to a truncated region obtained by truncating and deleting the functional region fragment of the full-length GM-CSF protein. The functional region fragment includes a signal peptid fragment, an A helix fragment, an exon 1 fragment, a C helix fragment and an exon 2 fragment. The full-length structure of the GM-CSF protein is shown in Figure 2 .

[0050] Based on the above modification means, the full-length GM-CSF protein is prepared into five short peptides containing different truncated fragments, specifically including:

[0051] GM-CSF first truncated region: only contains the A helix fragment region of GM-CSF, denoted as X1.

[0052] GM-CSF second truncated region: contains the exon 1 fragment region and the C helix fragment region of GM-CSF, denoted as X2.

[0053] GM-CSF third truncated region: only contains the exon 2 fragment region of GM-CSF, denoted as X3.

[0054] GM-CSF fourth truncated region: contains the A helix, C helix and exon 2 fragment regions of GM-CSF, denoted as X4.

[0055] GM-CSF fifth truncated region: contains the A helix, exon 1 and C helix fragment regions of GM-CSF, denoted as X5.

[0056] The purpose of the present application is to explore the influence of the GM-CSF second truncated region on CAR and the functional improvement.

[0057] The sequence information involved in the present application is as follows.

[0058] Table 1

[0059]

[0060] Example 1

[0061] Construction of novel CAR expression vector and virus packaging

[0062] 1.1 Construction of lentiviral expression vector of novel CAR vector

[0063] A lentiviral expression vector pWPXLD-CAR-P2A-X2 expressing novel CAR was constructed using the method of molecular cloning, and the gene fragment of the expression vector contained a CD8 signal peptide, a SCFV, a hinge, a human CD8 transmembrane region (TM), a human 4-1BB costimulatory signal region, a human CD3 zeta signal domain, a P2A, and a fragment (X2), as shown in Figure 1 . Wherein, X2 only contains exon 1 and C helix region of GM-CSF, and does not contain a signal peptide, that is, it can enhance the function of CAR intracellularly.

[0064] 1.2 Packaging of lentivirus of novel CAR

[0065] The 293T cells in good growth state were trypsinized, and were subcultured at a density of 4x10 6 cells / dish. When the cell confluence reached 80%, the CAR vector was used as the core plasmid, psPAX2 and pMD2.G were used as the auxiliary plasmid, and the calcium phosphate transfection technology was used for transfection. After 8 hours of transfection, the culture medium containing calcium phosphate precipitate was discarded, and the cell culture supernatant was collected at 48 and 72 hours after transfection, centrifuged at 2000 rpm for 10 minutes, and the cell precipitate was removed. A 0.22 μm disposable needle filter (PES membrane) was used to remove cell debris.

[0066] Example 2

[0067] CAR positive rate detection.

[0068] The T cells were transduced with the lentivirus in Example 1 to obtain novel CAR-T cells, the target protein expressing His tag was used as a primary antibody, and the His antibody coupled with fluorescein APC was used as a secondary antibody to detect the CAR positive rate. The conventional CAR was a traditional second-generation CAR-T, and the CAR-GM was a CAR containing full-length GM. The results are shown in Figure 3 . The results show that the positive rate of CAR-X2-T is the highest, reaching 65.35%; the positive rates of the traditional second-generation CAR-T and the CAR-T containing full-length GM as a control are about 55%.

[0069] Example 3

[0070] This example detects the proliferation ability of the CAR-X2-T cells synthesized in Example 1.

[0071] The same number of T cells were transduced with the lentivirus in Example 1 to obtain the new CAR-X2-T cells, and the number of CAR-X2-T cells was detected using a cell counter 3 days later to calculate the expansion fold, and the results are shown in Figure 4 The results show that the cell proliferation number of the conventional CAR-T is the lowest; the cell proliferation number of the CAR-GM-T is more than that of the conventional CAR-T; and the proliferation number of the CAR-X2-T cells is the largest. This indicates that the CAR retaining only part of the functional region of GM-CSF not only retains the transfection rate comparable to that of the conventional CAR and the CAR-GM, but also has the largest proliferation number in the same time and reduces the sequence length of the overexpressed gene.

[0072] Example 4

[0073] This example detects the verification of the CAR-X2-T cells synthesized in Example 1 for killing tumor cells.

[0074] SK-OV3 cells (human ovarian cancer cells) positive for the CAR target were used as target cells, and CAR-T cells were used as effector cells. The two kinds of cells were co-cultured at an effector-to-target ratio of 5:1, and the killing of SK-OV3 cells by CAR-X2-T cells was monitored.

[0075] As shown in Figure 5 , the killing ability of CAR-X2-T cells on tumor cells is higher than that of conventional CAR and is comparable to that of CAR-GM.

[0076] The above experimental results show that after truncating the full-length GM-CSF protein, the CAR prepared from the specific truncated functional region has a similar killing ability on tumor cells to the CAR prepared from the full-length GM-CSF protein, but has a higher positive expression rate and higher cell proliferation ability than the CAR prepared from the full-length GM-CSF protein.

[0077] Example 5

[0078] NK cells were further transduced with the lentivirus in Example 1 to obtain the new CAR-X2-NK cells. SK-OV3 cells positive for the CAR target were used as target cells, and CAR-X2-NK cells were used as effector cells. The two kinds of cells were co-cultured at an effector-to-target ratio of 5:1, and the killing of SK-OV3 cells by CAR-X2-NK cells was monitored.

[0079] The killing efficiency of the killing curve was analyzed for 30 hours, and the results show that the killing ability of CAR-X2-NK cells on tumor cells is higher than that of conventional CAR-NK as a whole, proving that the modification of CAR-X2 structure on NK cells is successful Figure 6 .

[0080] Further, the supernatant after co-culturing CAR-X2-NK cells with tumor cells for 24 hours was collected, and the IFN-γ and TNF-α in the supernatant were analyzed. The results showed that the IFN-γ and TNF-α in the supernatant after co-culturing CAR-X2-NK with target cells were significantly increased Figure 7 , and the specific results are as follows.

[0081] Table 2. CAR-NK cell detection results

[0082]

[0083] The results showed that, compared with the control group, CAR-X2-NK significantly increased the expression of IFN-γ and TNF-α in the co-culture supernatant. CAR-NK cells in the control group almost did not express TNF-α.

[0084] It should be noted that in this paper, the term "including", "containing" or any other variant thereof is intended to cover non-exclusive inclusion, so that the process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or includes elements inherent to such process, method, article or device. Without more limitations, the element defined by the statement "including a" does not exclude the presence of other identical elements in the process, method, article or device including the element.

[0085] The embodiments of the present application are described above in combination with the drawings, but the present application is not limited to the above specific embodiments, and the above specific embodiments are only illustrative and not limiting. Those skilled in the art can make many forms under the inspiration of the present application without departing from the purpose of the present application and the scope protected by the claims, and these are all within the protection of the present application.

Claims

1. A CAR-NK cell expressing a truncated functional region fragment 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 truncated functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the truncated functional region fragment of the GM-CSF protein includes an exon 1 functional region fragment and a Chelix functional region fragment, and the nucleotide sequence of the truncated functional region fragment of the GM-CSF protein is shown in SEQ ID NO.1; the 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 of 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 novel chimeric antigen receptor structure is also linked to the EF1-α promoter sequence.

3. The use of the CAR-NK cells according to claim 1 or 2 in the preparation of an immune enhancer for adjuvant tumor therapy.

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