Immunologically engineered cells expressing a functional region of exon 2 of gm-csf and uses thereof

Abstract

The application belongs to the technical field of biology and specifically relates to an immune engineering cell expressing a GM-CSF exon 2 functional region 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 exon 2 functional region fragment and a chimeric antigen receptor. The application also provides a CAR-T cell also expressing a GM-CSF exon 2 functional region fragment. The application proposes a novel chimeric antigen receptor structure containing only a functional region fragment of a full-length GM-CSF, which not only retains the functions of conventional CAR and CAR-GM, but also achieves improved functions.

Description

[0001] Priority Application

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

CN2023115953752

A CAR-T containing GM-CSF 3rd truncated region and preparation method and application

CN2023115953911

A CAR-T containing GM-CSF 4th truncated region and preparation method and application

[0003] The present application belongs to the field of biotechnology, and specifically relates to an immune engineering cell expressing the exon 2 functional region of GM-CSF 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) signal transduction 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 did not achieve satisfactory results, 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] After that, CAR-T related technologies have been continuously innovated, but among the 939 immune cell therapy trials initiated since 1993, only about half are for solid tumors. However, 90% of the global cancer incidence rate is solid tumors. Unfortunately, compared to the good effect shown on hematological tumors, the effect of most reported CAR-T cell treatments for solid tumors has always been difficult to effectively break through at some key stages. Therefore, there is still a need for technological 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 the inhibitory signal and the activation signal in the NK cell is biased towards activation, the NK cell can form a synapse with the target cell, thereby releasing effector granules to lyse the target cell 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 because 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 CD3 zeta, so the traditional CAR molecule design that works on CAR-T may not necessarily work well in NK cells.

[0008] In summary, it is necessary to improve the prior art. SUMMARY

[0009] The purpose of the present application is to provide an immune engineering cell expressing the exon 2 functional region of GM-CSF and its application, and the specific technical solutions are as follows.

[0010] A CAR-NK cell expressing the exon 2 functional region of GM-CSF, the CAR-NK cell expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises an exon 2 functional region fragment of GM-CSF protein and a chimeric antigen receptor; the exon 2 functional region fragment is connected with 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 costimulatory signal region and a human CD3 zeta signal domain.

[0011] As a preferred, the nucleotide sequence of the exon 2 functional region fragment is shown as SEQ ID NO. 1.

[0012] Further, the novel chimeric antigen receptor structure is also connected with an EF1-alpha promoter sequence.

[0013] As a preferred, the nucleotide sequence of the chimeric antigen receptor expressed by the CAR-T cell is shown as SEQ ID NO. 3.

[0014] A CAR-T cell expressing a GM-CSF exon 2 functional region, the CAR-T cell expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises a GM-CSF protein exon 2 functional region fragment and a chimeric antigen receptor; the exon 2 functional region fragment is connected with 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 costimulatory signal region and a human CD3 zeta signal domain.

[0015] As a preferred, the nucleotide sequence of the exon 2 functional region fragment is shown as SEQ ID NO. 1.

[0016] Further, the novel chimeric antigen receptor structure is also connected with an EF1-alpha promoter sequence.

[0017] As a preferred, the nucleotide sequence of the chimeric antigen receptor expressed by the CAR-T cell is shown as SEQ ID NO. 3.

[0018] The application can also provide the use of the above-mentioned CAR-NK cell in the preparation of a drug for treating solid tumors.

[0019] Compared with CAR-T cells, CAR-NK cells have some significant advantages, including: (1) cytokine release syndrome and neurotoxicity are less. (2) CAR-NK cells can kill cancer cells through two pathways of CAR-dependent and CAR-independent. In addition, NK cells can kill tumor cells through CD16-mediated ADCC. Therefore, CAR-NK cells have broad-spectrum and high-efficiency tumor killing activity. (3) Allogeneic reinfusion therapy can be achieved.

[0020] The use of the above-mentioned CAR-T cell in the preparation of a drug for treating solid tumors.

[0021] Further, the tumor comprises a hematological tumor, liver cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, ovarian cancer, glioma, pancreatic cancer, sarcoma or glioblastoma.

[0022] The CAR-T cell or the CAR-NK cell can enhance the killing effect on tumor cells by expressing the exon 2 of the GM-CSF protein.

[0023] The CAR-NK cell is used for preparing a TNF-α promoter.

[0024] As a preferred, the CAR-NK cell can secrete more TNF-α as a TNF-α promoter.

[0025] The CAR-NK cell is used for preparing an IFN-γ promoter.

[0026] As a preferred, the CAR-NK cell can secrete more IFN-γ as an IFN-γ promoter.

[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] The application further provides a lentiviral vector of a CAR expression vector, which comprises a nucleic acid encoding a chimeric antigen receptor and a nucleic acid encoding an exon 2 functional region of GM-CSF; the nucleic acid encoding the chimeric antigen receptor is connected with the nucleic acid encoding the exon 2 functional region of GM-CSF through a sequence encoding P2A; the chimeric antigen receptor comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB costimulatory signal region and a human CD3 zeta signal domain; and the lentivirus comprises pWPXLD, psPAX2 and pMD2.G.

[0029] The application can further comprise a preparation method of the CAR or a preparation method of the CAR-T or the CAR-NK.

[0030] Beneficial technical effects:

[0031] In one aspect, the present application constructs an immune engineering cell expressing a unique functional region exon 2 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 application only contains one functional region 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 improved proliferation ability of CAR-T cells. The proliferation ability of CAR-X3-T cells of the present application is superior to that of conventional CAR-T cells and CAR-GM-T cells. Obviously, the yield of CAR-X3-T cells of the present application is higher and the production cost is lower when used for industrial production.

[0032] In another aspect, the present application experimentally proves the influence of reducing the number of functional regions (i.e. the influence of containing only a unique functional region and containing other functional regions in addition to the unique functional region) on the results under the premise of containing the same truncated functional region of GM-CSF. The results prove that the CAR containing only the unique functional region exon 2 screened by the present application, as the modification of containing the least functional region fragment of the full-length GM-CSF, not only retains the function comparable to that of conventional CAR and CAR-GM, but also achieves improved function. Specifically, the killing ability of CAR-X3-T cells prepared by the present application on tumors is significantly stronger than that of conventional CAR-T cells as a whole, and is comparable to that of GM-CSF full-length CAR-T cells, and is better than that of CAR-X4-T cells containing 3 functional region fragments. More cytokine secretion can be detected in the supernatant of CAR-X3-NK cells prepared by the present application co-cultured with tumor cells. Therefore, the CAR-X3-NK cells or CAR-X3-T cells of the present 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, which needs to be experimentally verified. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present 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 present application, and those skilled in the art can obtain other drawings according to these drawings without paying creative labor.

[0035] Figure 1 Figure 1 is a schematic diagram of the structure of a novel CAR expression vector of the present application;

[0036] Figure 2 Figure 2 is a schematic diagram of the functional region of the full-length GM-CSF protein;

[0037] Figure 3 Figure 3 is a graph showing the results of CAR positive rate detection of CAR-X3-T cells in one embodiment of the present application;

[0038] Figure 4 Figure 4 is a graph showing the proliferation results of CAR-X3-T cells and control cells (CAR-X4-T) in one embodiment of the present application;

[0039] Figure 5 Figure 5 shows that the killing efficiency of CAR-X3-T cells and control cells (CAR-X4-T) on tumor cells is higher than that of conventional CAR-T cells;

[0040] Figure 6 Figure 6 shows that the TNF-α secreted by CAR-X3-NK cells is 59.6 times that of conventional CAR-NK cells, and the IFN-γ is 1.7 times that of conventional CAR-NK cells;

[0041] Figure 7 Figure 7 shows the killing curve of CAR-X3-NK cells on tumor cells. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme of the embodiments of the present application will be described clearly and completely below with reference to the drawings of the embodiments of the present application. Obviously, the described embodiments are part 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 those skilled in the art without creative labor fall within the scope of protection 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, etc.

[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 a format indicating that the embodiments can be combined with one another. It is to be understood that, unless otherwise indicated herein, these combinations are not necessarily mutually exclusive, and a single aspect of the application can be claimed in a dependent manner as multiple alternatives unless otherwise indicated. Additionally, it is to be understood that the features can be combined in any suitable manner without departing from the scope of the application.

[0047] The "new chimeric antigen receptor structure" of the present application refers to one of the functional region fragments (X3) 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.

[0048] The "GM-CSF truncated region" of the present application refers to a truncated region obtained by truncating and deleting the functional region fragments of the full-length GM-CSF protein. The functional region fragments include 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 .

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

[0050] The first truncated region of GM-CSF mainly contains the A helix region of GM-CSF, denoted as X1.

[0051] The second truncated region of GM-CSF mainly contains the exon 1 and C helix regions of GM-CSF, denoted as X2.

[0052] The third truncated region of GM-CSF mainly contains the exon 2 region of GM-CSF, denoted as X3.

[0053] The fourth truncated region of GM-CSF mainly contains the A helix, C helix, and exon 2 regions of GM-CSF, denoted as X4.

[0054] The fifth truncated region of GM-CSF mainly contains the A helix, exon 1 and C helix regions of GM-CSF, which is denoted as X5.

[0055] In one aspect, the present application aims to explore the improvement of CAR function by truncating the full-length protein of GM-CSF.

[0056] In another application, the present application aims to explore the minimum truncated unit that can maintain the function of CAR and has a certain functional improvement under the premise of truncating the full-length protein of GM-CSF.

[0057] The sequence information involved in the present application is shown in Table 1.

[0058] Table 1

[0059]

[0060] Example 1

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

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

[0063] A lentiviral expression vector pWPXLD-CAR-P2A-X3 expressing a novel CAR was constructed using a molecular cloning method, and the gene fragment of the expression vector contains a CD8 signal peptide, a SCFV, a hinge region, a human CD8 transmembrane region (TM), a human 4-1BB costimulatory signal region, a human CD3 ζ signal domain, a P2A, and a fragment (X3), as shown in Figure 1 . Among them, X3 mainly contains the exon 2 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 a lentivirus of a novel CAR

[0065] Good 293T cells were taken, the cells were trypsinized, and the cells were passaged at a density of 4×10 6 cells / dish. When the confluence of the cells 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 technique 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, i.e., the virus stock solution, centrifuged at 2000 rpm for 10 minutes, and the cell precipitate was removed, and a 0.22 μm disposable needle filter (PES membrane) was used to remove cell debris.

[0066] Example 2

[0067] CAR positive rate detection

[0068] Using the lentivirus in Example 1 to transduce T cells, new CAR-T cells were obtained, and the CAR positive rate was detected using a target protein expressing a His tag as a primary antibody and a His antibody coupled with fluorescein APC as a secondary antibody. The conventional CAR was a traditional second-generation CAR-T, CAR-GM was a CAR containing a full-length GM, and CAR-X3 was a CAR containing a fragment X3. The results are shown in Figure 3 The results show that the positive rate of CAR-X3-T reached 58%, which is not much different from that of traditional CAR-T cells and CAR-GM-T cells, indicating that a CAR containing only one functional region, exon 2, still retains a transfection rate comparable to that of traditional CAR and CAR-GM.

[0069] Example 3

[0070] This example detects the proliferation ability of the CAR-X3-T cells synthesized in Example 2.

[0071] Using the same number of T cells transduced by the lentivirus in Example 1, new CAR-X3-T cells were obtained, and the number of CAR-X3-T cells was detected using a cell counter after 3 days, and the expansion fold was calculated. The results are shown in Figure 4 The results show that the cell proliferation fold of traditional CAR-T is the lowest; the cell proliferation fold of CAR-GM-T is higher than that of traditional CAR-T; and the cell proliferation fold of CAR-X3-T is the highest. This indicates that a CAR containing only one functional region, exon 2, not only retains a transfection rate comparable to that of traditional CAR and CAR-GM, but also has the highest proliferation fold in the same time.

[0072] To further explore the effect of the truncated functional region of GM-CSF on cell proliferation, the following control experiments are also provided in this example.

[0073] Using a similar method as in Example 1, the same number of T cells were transduced to construct CAR-X4-T (control cells), wherein the CAR structure of CAR-X4-T and CAR-X3-T is consistent. The X4 fragment contains three functional regions of the A helix fragment, the C helix fragment, and the exon 2 fragment of GM-CSF, and the specific example is as follows.

[0074] Table 2

[0075]

[0076] After 3 days, the proliferation of CAR-X3-T cells and CAR-X4-T cells was detected using a cell counter, and the expansion fold was calculated. See Figure 4The results show that the proliferation ability of the CAR-X-T cells prepared by truncating the full-length GM-CSF to retain the necessary functional regions is stronger than that of the CAR-T cells prepared by the full-length GM-CSF. However, based on the functional region of X4, further deletion of the functional region obtains X3 containing only a functional region fragment, which has a smaller effect on the proliferation of CAR-T cells than CAR-X4-T, but is stronger than the cell proliferation ability of the conventional CAR-T and CAR-GM-T in general. This shows that the CAR containing only the unique functional region exon 2 proposed in the application, as the modification containing the least functional region fragment in the full-length GM-CSF, not only retains the function comparable to the conventional CAR and CAR-GM, but also achieves improved function.

[0077] The nucleotide sequence of X4 in the CAR-X4-T cells involved in the present embodiment is as follows.

[0078] Table 3

[0079]

[0080] Example 4

[0081] The present embodiment detects the verification of the killing of tumor cells by the CAR-X3-T cells synthesized in Example 2.

[0082] SK-OV3 cells (human ovarian cancer cells) positive for the CAR target are used as target cells, and CAR-X3-T cells are used as effector cells. The two kinds of cells are co-cultured at an effector-to-target ratio of 5:1, and the killing of SK-OV3 cells by CAR-X3-T cells is monitored.

[0083] As shown in Figure 5 , 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 from about 6 h, and this killing advantage is more significant after about 10 h. The killing efficiency of the killing curve for 40 h is analyzed, and the results show that the killing efficiency of CAR-X3-T cells on tumor cells is comparable to that of CAR-GM-T cells.

[0084] To further explore the effect of the truncated functional region of GM-CSF on cell proliferation, the present embodiment also performs a control experiment using CAR-X4-T cells as control cells. The results show that Figure 5 , there is no obvious difference in the killing of tumor cells by CAR-X3-T cells and CAR-X4-T cells.

[0085] Example 5

[0086] Further, the NK cells were transduced by the lentivirus in Example 1 to obtain the novel CAR-X3-NK cells. The supernatant after the CAR-X3-NK cells were co-cultured with the tumor cells for 24 hours was collected, and the IFN-γ and TNF-α in the supernatant were analyzed. The IFN-γ and TNF-α in the supernatant after the CAR-NK cells were co-cultured with the target cells were significantly increased Figure 6 , and the specific results are as follows.

[0087] Table 4: Detection results of the CAR-X3-NK cells.

[0088]

[0089] The results show that the TNF-α secreted by the CAR-X3-NK cells is 59.6 times that of the conventional CAR-NK cells; the CAR-NK cells in the control group hardly express TNF-α. The IFN-γ is 1.7 times that of the conventional CAR-NK cells. It is proved that X3, as a modified selection of the minimum functional region fragment of GM-CSF, plays the function of an immunomodulatory factor, and significantly increases the expression of IFN-γ and TNF-α of the CAR-NK cells compared with the control group.

[0090] Further, based on the novel CAR-X3-NK cells obtained above, the SK-OV3 cells positive to the CAR target were used as target cells, and the CAR-X3-NK cells were used as effector cells. The two kinds of cells were co-cultured according to the effector-target ratio of 5:1, and the killing of the CAR-X3-NK cells on the SK-OV3 cells was monitored.

[0091] As shown in Figure 7 , the killing efficiency was analyzed for 30 hours, and the results show that the killing ability of the CAR-X3-NK cells on the tumor cells is slightly weaker than that of the conventional CAR-NK cells.

[0092] The above results show that the CAR-NK cells containing the modified CAR-NK cells of the unique functional region exon 2 of GM-CSF protein screened by the application have stronger cytokine secretion ability than the conventional CAR-NK cells, but the killing effect on the tumor cells is not necessarily better than that of the conventional CAR-NK cells. Therefore, the CAR-NK cells of the application have the potential to prepare IFN-γ promoters and TNF-α promoters.

[0093] It should be noted that, in this document, the terms "comprising", "comprises" or any other variation thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can also include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element preceded by "comprises a" does not, without more limitations, foreclose the existence of additional identical elements in the process, method, article, or apparatus that comprises the recited element.

[0094] The embodiments of the present application are described above with reference to the accompanying drawings, but the present application is not limited to the above-described specific embodiments, and the above-described specific embodiments are merely illustrative, not restrictive, and a person of ordinary skill 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.

Claims

1. A CAR-NK cell expressing a functional region of exon 2 of GM-CSF, characterized in that, The CAR-NK cell expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure is an EF1-alpha promoter sequence, a chimeric antigen receptor, a P2A sequence and an exon 2 functional region fragment in sequence; the exon 2 functional region fragment is connected with the chimeric antigen receptor through the P2A sequence; the chimeric antigen receptor comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB costimulatory signal region and a human CD3 zeta signal domain; the nucleotide sequence of the exon 2 functional region fragment is shown as SEQ ID NO. 1; and the nucleotide sequence of the chimeric antigen receptor expressed by the CAR-NK cell is shown as SEQ ID NO. 3.

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