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

Abstract

The present application belongs to the field of biotechnology, and particularly relates to immune engineering cells expressing Ahelix functional region of GM-CSF and application. The present application first provides a CAR-NK cell, which expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises an Ahelix functional region fragment of GM-CSF protein and a chimeric antigen receptor. The present application also provides a CAR-T cell also expressing an Ahelix functional region fragment of GM-CSF. The present application proposes a novel chimeric antigen receptor structure only containing the least functional region fragment of full-length GM-CSF, which not only retains the function equivalent to that of conventional CAR and full-length GM-CSF CAR, but also achieves improved function.

Description

[0001] Priority application

[0002] This application claims priority to Chinese invention patent application No. CN2023115952393, filed November 24, 2023, entitled “A CAR-T containing the first truncated region of GM-CSF, a preparation method and application thereof”; Chinese invention patent application No. CN2023115817245, filed November 24, 2023, entitled “A CAR-T containing the second truncated region of GM-CSF, a preparation method and application thereof”; and Chinese invention patent application No. CN2023115954083, filed November 24, 2023, entitled “A CAR-T containing the fifth truncated region of GM-CSF, a preparation method and application thereof”, all of which are incorporated herein by reference in their entirety. Technical Field

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

[0004] Chimeric antigen receptors (CARs) are artificial receptor molecules manufactured using genetic engineering technology. They can endow immune effector cells (such as T cells and NK cells) with specificity for a target antigen epitope, thereby enhancing the function of lymphocytes in recognizing antigen signals and activating. First-generation CARs only contain CD3ξ (containing 3 ITAMs) and FcRγ (containing 2 ITAMs) signaling domains, providing only the first signal for T cell activation. Early clinical trials of first-generation CARs showed that they could not effectively maintain T cell proliferation and activation, failing to achieve satisfactory results, indicating that first-generation CAR-T cells may lack sufficient activation signals to maintain T cell proliferation and effective anti-tumor effects. Based on first-generation CAR-T, second-generation CAR-T was developed. Second-generation CAR-T cells have shown enhanced in vivo expansion and persistence, and their efficacy has been confirmed in clinical trials.

[0005] Since then, CAR-T related technologies have been continuously innovated, but of the 939 immunotherapy trials initiated since 1993, only about half have targeted solid tumors. However, solid tumors account for 90% of global cancer incidence. Unfortunately, compared to the good efficacy shown in hematologic malignancies, most reported efficacy of CAR-T cell therapy for solid tumors has consistently failed to achieve significant breakthroughs at certain key stages. Therefore, technological innovation is still needed in this field.

[0006] NK cells are important members of the innate immune system, mainly found in blood and lymphatic organs. They do not require pre-sensitization and are not restricted by the major histocompatibility complex (MHC), enabling them to rapidly and directly kill target cells and exhibiting broad-spectrum anti-tumor activity. NK cells kill target cells in various ways. Once the balance between inhibitory and activating signals within NK cells shifts towards activation, NK cells can form synapses with target cells, releasing effector granules to lyse the target cells and producing effector cytokines. Expressing chimeric antigen receptors (CARs) on the surface of NK cells can significantly enhance the anti-cancer effect of immune cells. Currently, the scientific community believes that CAR-NK cells have some significant advantages compared to CAR-T cells. However, the preparation of CAR-NK cells with tumor-killing efficacy is challenging, mainly due to the difficulty in genetically modifying NK cells because of their natural antiviral function.

[0007] The patent, CN114934071A, entitled "A CAR Vector Expressing an Immunomodulatory Factor and Its Application," discloses a CAR-T cell expressing a virally transduced chimeric antigen receptor and an immunomodulatory factor, the full-length granulocyte-macrophage colony-stimulating factor (GM-CSF). NK cell activation receptors include natural cytotoxic receptors (NCRs) such as NKG2D, CD16 (FcgRIIIa), FasL, and tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL), as well as co-stimulatory receptors such as LFA-1, CD244 (2B4), and CD137 (41BB). However, the intracellular signaling activation domains of CAR-T cells are mainly 41BB, CD28, and CD3ζ. Therefore, traditional CAR molecule designs that are effective for CAR-T cells may not necessarily exert good activation effects in NK cells.

[0008] In conclusion, it is necessary to make improvements to the existing technology. Summary of the Invention

[0009] The purpose of this invention is to provide immune-engineered cells that express only the Ahelix functional region of GM-CSF and their applications. The specific technical solution is as follows.

[0010] A CAR-NK cell expressing the Ahelix functional region of GM-CSF, wherein the CAR-NK cell expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises an Ahelix functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the Ahelix functional region fragment and the chimeric antigen receptor are linked 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 signaling region, and a human CD3ζ signaling domain.

[0011] As a preferred embodiment, the nucleotide sequence of the Ahelix functional region fragment is shown in SEQ ID NO.1.

[0012] Furthermore, the novel chimeric antigen receptor structure is also linked to an EF1-α promoter sequence.

[0013] As a preferred embodiment, the nucleotide sequence of the chimeric antigen receptor expressed by the CAR-NK cells is shown in SEQ ID NO. 3.

[0014] A CAR-T cell expressing the Ahelix functional region of GM-CSF, wherein the CAR-T cell expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises an Ahelix functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the Ahelix functional region fragment and the chimeric antigen receptor are linked 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.

[0015] As a preferred embodiment, the nucleotide sequence of the Ahelix functional region fragment is shown in SEQ ID NO.1.

[0016] Furthermore, the novel chimeric antigen receptor structure is also linked to an EF1-α promoter sequence.

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

[0018] The above-mentioned CAR-NK cells are used in the preparation of drugs for treating solid tumors.

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

[0020] The above-mentioned application of CAR-T cells in the preparation of drugs for treating solid tumors.

[0021] The aforementioned CAR-NK cells or CAR-T cells can enhance their killing effect on tumor cells by expressing GM-CSF protein in A helix.

[0022] The antigen-binding domain in the CAR provided by this invention can target CD19, BCMA, HER2, Claudin18.2, Mesothelin, GPC3, or GD2, etc.

[0023] Furthermore, the tumors include hematologic malignancies, liver cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, ovarian cancer, glioma, pancreatic cancer, sarcoma, or glioblastoma.

[0024] The present invention may also include a method for preparing the above-mentioned CAR, or a method for preparing the above-mentioned CAR-T cells or CAR-NK cells.

[0025] Beneficial technical effects:

[0026] On one hand, this invention constructs an immunoengineered cell expressing a single functional region of GM-CSF, which expresses a novel chimeric antigen receptor structure. Compared to CARs containing the full-length GM-CSF protein, the novel chimeric antigen receptor structure of this invention contains only one functional region of the full-length GM-CSF protein, reducing the sequence length of the overexpressed gene without affecting CAR expression and function. On the contrary, since this truncated functional region does not contain a signal peptide, it can function intracellularly, resulting in enhanced CAR-T cell proliferation. The proliferation capacity of the CAR-X1-T cells of this invention is superior to that of conventional CAR-T cells and CAR-GM-T cells. Obviously, the CAR-X1-T cells of this invention have higher yields and lower production costs when used for industrial production.

[0027] On the other hand, the experiments of this invention confirmed the impact of reducing the number of functional regions on the results, provided that different GM-CSF truncated functional regions are included. The results confirmed that the CAR proposed in this invention, containing only the single functional region A helix, as a modification containing only the minimum functional region fragment of full-length GM-CSF, not only retains functions comparable to traditional CARs and CAR-GM, but also achieves improved functions. Specifically, the CAR-X1-T cells prepared in this invention exhibit better overall tumor-killing ability than conventional CAR-T and full-length GM-CSF CAR-T; more cytokine secretion can be detected in the supernatant of CAR-X1-NK cells co-cultured with tumor cells. Therefore, the CAR-X1-NK cells or CAR-X1-T cells of this invention have the potential to become adjuvant therapy products and play a role in the preparation of anti-tumor therapeutic drugs.

[0028] Finally, due to the differences between NK cell modification and T cell modification, CAR structures that are expressed and function on CAR-T cells may not necessarily have the same function on CAR-NK cells, which requires experimental verification. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0030] Figure 1 This is a construction diagram of the novel CAR structure expression vector of the present invention;

[0031] Figure 2 This is a schematic diagram of the functional region of the full-length CM-CSF protein;

[0032] Figure 3 This is a graph showing the results of CAR-X1-T cell CAR positivity rate detection in one embodiment of the present invention;

[0033] Figure 4 This is a diagram showing the proliferation results of CAR-X1-T cells and control cells (CAR-X2-T and CAR-X5-T) in one embodiment of the present invention;

[0034] Figure 5 In one embodiment of the present invention, CAR-X1-T cells showed a higher tumor cell killing efficiency than the control group CAR-T cells;

[0035] Figure 6 In one embodiment of the present invention, the overall killing ability of CAR-X1-NK cells against tumor cells is higher than that of conventional CAR-T cells;

[0036] Figure 7 In one embodiment of the present invention, CAR-X1-NK cells secreted higher levels of TNF-α and IFN-γ compared to conventional CAR-NK cells. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0038] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0039] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0040] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0041] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0042] Definition of noun:

[0043] The "novel chimeric antigen receptor structure" described in this invention refers to a chimeric antigen receptor (CAR) and a functional region fragment (X1) of the GM-CSF protein linked to the chimeric antigen receptor via 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 signaling region, and a human CD3ζ signaling domain.

[0044] The "GM-CSF truncated region" described in this invention refers to a truncated region obtained by modifying the full-length GM-CSF protein by cutting off and deleting functional region fragments; wherein, the functional region fragments include the signal peptid fragment, the A helix fragment, the exon 1 fragment, the C helix fragment, and the exon 2 fragment. The full-length structure of the GM-CSF protein is as follows. Figure 2 As shown.

[0045] Based on the above modification methods, the full-length GM-CSF protein was prepared into 5 short peptides containing different truncated fragments, specifically including:

[0046] GM-CSF 1st truncated region: A helix fragment region containing only GM-CSF, denoted by X1.

[0047] GM-CSF 2nd truncated region: Contains the exon 1 fragment region and the C helix fragment region of GM-CSF, denoted as X2.

[0048] GM-CSF 3rd truncated region: The region containing only the exon 2 fragment of GM-CSF, denoted as X3.

[0049] GM-CSF 4th truncated region: Contains the A helix, C helix and exon 2 fragment regions of GM-CSF, denoted as X4.

[0050] GM-CSF 5th truncated region: Contains the A helix, exon 1 and C helix fragment regions of GM-CSF, denoted as X5.

[0051] On the one hand, the purpose of this invention is to explore the improvement of CAR function by truncating the full-length GM-CSF protein.

[0052] Another invention aims to explore the minimum number of truncated units that can maintain the function of CAR and have a certain functional improvement while truncating the full-length GM-CSF protein.

[0053] The sequence information involved in this invention is shown in Table 1.

[0054] Table 1

[0055]

[0056] Example 1

[0057] Construction of novel CAR expression vectors and viral packaging.

[0058] 1.1 Construction of lentiviral expression vectors for novel CAR vectors

[0059] A novel lentiviral expression vector, pWPXLD-CAR-P2A-X1, was constructed using molecular cloning methods. This vector contains the following gene fragments: CD8 signal peptide, ScFV, hinge region, human CD8 transmembrane region (TM), human 4-1BB co-stimulatory signaling region, human CD3 ζ signaling domain, P2A, and fragment (X1). Figure 1 As shown in the figure. Among them, X1 contains only the A helix region of GM-CSF and does not contain a signal peptide, meaning that it can enhance the function of CAR intracellularly.

[0060] 1.2 Packaging of novel CAR lentiviruses

[0061] Take 293T cells in good growth condition, digest the cells with trypsin, and then... 6The cells were passaged at a density of cells / plate until 80% confluence was achieved. Transfection was then performed using a CAR vector as the core plasmid and psPAX2 and pMD2.G as helper plasmids via calcium phosphate transfection. Eight hours after transfection, the medium containing the calcium phosphate precipitate was discarded. The cell culture supernatant was collected at 48 and 72 hours after transfection as the viral stock solution. The cells were centrifuged at 2000 rpm for 10 minutes to remove the cell pellet, and cell debris was removed using a 0.22 μm disposable syringe filter (PES membrane).

[0062] Example 2

[0063] CAR positivity rate detection.

[0064] Novel CAR-T cells were obtained by transducing T cells with the lentivirus described in Example 1. The CAR positivity rate was detected using a His-tagged target protein as the primary antibody and a His antibody conjugated with fluorescein APC as the secondary antibody. Conventional CAR refers to traditional second-generation CAR-T cells, CAR-GM is a CAR containing the full-length GM-CSF, and CAR-X1 is a CAR containing only the X1 fragment. Results are as follows... Figure 3 As shown in the figure. The results showed that the positivity rate of CAR-X1-T reached 50%, which was not much different from that of traditional CAR and CAR-GM, indicating that the transfection efficiency of CAR containing only one functional region, A helix, was comparable to that of traditional CAR and CAR-GM.

[0065] Example 3

[0066] This embodiment tests the proliferation ability of CAR-X1-T cells synthesized in Example 1.

[0067] The same number of T cells were transduced using the lentivirus from Example 1 to obtain novel CAR-X1-T cells. Three days later, the number of CAR-X1-T cells was detected using a cell counter, and the fold increase was calculated. The results are as follows: Figure 4 As shown in the figure, the results indicated that traditional CAR-T cells had the lowest cell proliferation rate; CAR-GM-T cells had a higher cell proliferation rate than traditional CAR-T cells; and CAR-X1-T cells had the highest proliferation rate. This suggests that CARs containing only the unique functional region A helix not only maintain a transfection rate comparable to traditional CARs and CAR-GM, but also produce CAR-X1-T cells with the highest proliferation rate in the same time period.

[0068] To further investigate the effect of GM-CSF truncated functional regions on cell proliferation, this embodiment also provides the following control experiment.

[0069] Using a method similar to that in Example 1, the same number of T cells were transduced to construct CAR-X2-T (Control 1) and CAR-X5-T (Control 2), respectively. The CAR structures of CAR-X2-T and CAR-X5-T are identical to those of CAR-X1-T. The X2 fragment contains two functional regions: the exon 1 fragment and the C helix fragment of GM-CSF; the X5 fragment contains three functional regions: the A helix fragment, the exon 1 fragment, and the C helix fragment of GM-CSF, as illustrated in the following example.

[0070] Table 2

[0071]

[0072] Three days later, the proliferation of CAR-X2-T cells and CAR-X5-T cells was detected using a cell counter, and the fold increase was calculated. (See attached image.) Figure 4 The results showed that truncating the full-length GM-CSF while retaining the necessary functional regions resulted in CAR-XT cells with stronger proliferative capacity than CAR-T cells prepared from full-length GM-CSF. However, removing one functional region from X5 to obtain X2 resulted in a smaller impact on CAR-T cell proliferation. Furthermore, removing another functional region from X2 to obtain X1 resulted in an impact on CAR-T cell proliferation comparable to that of X2, and overall, it exhibited stronger cell proliferation capacity compared to traditional CARs and CAR-GM. This indicates that the CAR proposed in this invention, containing only the single functional region A helix, as a modification containing only the minimum functional region fragments from full-length GM-CSF, not only retains functions comparable to traditional CARs and CAR-GM but also achieves improved functionality.

[0073] In this embodiment, CAR-X2-T and CAR-X5-T represent the second and fifth truncated regions of GM-CSF, respectively, and their nucleotide sequences are as follows.

[0074] Table 3

[0075]

[0076] Example 4

[0077] This embodiment verifies the killing of tumor cells by the CAR-X1-T cells synthesized in Example 1.

[0078] CAR-target-positive SK-OV3 cells (human ovarian cancer cells) were used as target cells, and CAR-T cells were used as effector cells. The two cell types were co-cultured at an effector-to-target ratio of 5:1, and the killing effect of CAR-X1-T cells on SK-OV3 cells was monitored.

[0079] like Figure 5 As shown, from approximately 5 hours onwards, CAR-X1-T cells exhibit a higher tumor-killing ability than conventional CAR and CAR-GM cells, and this advantage becomes even more pronounced after 10 hours. Analysis of the killing curve at 40 hours further demonstrates that the overall tumor-killing efficacy of CAR-X1-T cells is significantly better than that of conventional CAR-T and CAR-GM-T.

[0080] The above experimental results demonstrate that the CAR proposed in this invention, which contains only the unique functional region A helix, is a modified CAR containing only the fewest functional region fragments in the full-length GM-CSF. Compared with the control group, it kills tumor cells earlier and has a more significant tumor-killing effect.

[0081] Example 5

[0082] The lentivirus from Example 1 was used to transduce NK cells to obtain novel CAR-X1-NK cells. CAR-target positive SK-OV3 cells were used as target cells, and CAR-X1-NK cells were used as effector cells. The two cell types were co-cultured at an effector-to-target ratio of 5:1, and the killing effect of CAR-X1-NK cells was monitored.

[0083] like Figure 6 As shown, the killing efficacy of the killing curve after 30 hours was analyzed. The results showed that the killing ability of CAR-X1-NK cells against tumor cells was on par with that of conventional CAR, proving that the modification of NK cells by the CAR-X1 structure was successful.

[0084] Furthermore, the supernatant of CAR-X1-NK cells and tumor cells were collected after 24 hours of co-culture, 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 were significantly increased after co-culturing CAR-X1-NK cells with target cells. Figure 7 The specific results are shown in the table below.

[0085] Table 4 CAR-X1-NK cell detection results

[0086]

[0087] The results showed that CAR-X1-NK cells secreted 9.4 times more TNF-α than conventional CAR-NK cells, while CAR-NK cells in the control group expressed almost no TNF-α. CAR-X1-NK cells secreted 1.8 times more IFN-γ than conventional CAR-NK cells. This demonstrates that X1, as a modified selection of the minimum functional region fragment of GM-CSF, functions as an immunomodulatory factor, significantly increasing the expression of IFN-γ and TNF-α in CAR-NK cells compared to the control group.

[0088] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0089] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A CAR-NK cell expressing the Ahelix 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 Ahelix functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the Ahelix functional region fragment and the chimeric antigen receptor are linked 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 Ahelix functional region fragment is shown in SEQ ID NO.1; 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 a medicament for treating solid tumors.

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