Unmethylated CpG oligodeoxynucleotides and their applications

Abstract

The present invention provides an oligodeoxynucleotide of unmethylated cytosine-phosphate-guanine dinucleotide and B lymphocytes bound thereto, which can target migration to diseased lymphoid organs and inhibit B lymphocytes activated under pathological conditions, thereby having a targeted therapeutic effect against pathological damage caused by B lymphocyte activation.

Description

[Technical Field]

[0001] This invention discloses oligodeoxynucleotides belonging to the nucleic acid technology field. [Background technology]

[0002] Toll-like receptors (TLRs) are an important group of protein molecules involved in nonspecific immunity (innate immunity). In this context, human Toll-like receptor 9 (TLR9) is primarily involved with B lymphocytes and CD123 + TLR9 is expressed in DC cells, and activated B lymphocytes express it at higher levels than quiescent B lymphocytes. By recognizing pathogen-associated CpG ODNs, TLR9 mediates innate and adaptive immunity and is involved in a variety of physiological and pathological processes such as inflammation, immunity, infection, and allergy. TLR9 / MyD88 / NF-κB is one of the body's important defense pathways against disease. TLR9 agonists are used as adjuvant components in vaccines to enhance the immune response, and products such as the Heplisav hepatitis B vaccine and the COVID-19 vaccine have received marketing approval. At the same time, TLR9 is currently one of the most promising targets in tumor immunotherapy and has great potential in monotherapy and combination therapy with other antitumor therapies. As a type of innate immune-stimulating molecule, TLR agonists, when used in combination with checkpoint inhibitors, can activate the body's own immune system, enhance the antitumor activity of checkpoint inhibitors, and strengthen immunity against refractory tumor antigens. Most of these are currently in Phase I / II clinical trials.

[0003] However, an excessively strong immune response can cause damage to the body (such as cell and tissue damage or dysfunction), leading to the development or progression of autoimmune diseases. Currently, the main mechanisms for inducing autoimmune tissue damage are thought to be autoantibody-mediated and lymphocyte-mediated immune damage. In patients with systemic lupus erythematosus (SLE), localized or systemic lymphadenopathy is observed, mainly in the cervical, submandibular, and axillary lymph nodes. This suggests that B lymphocytes are abnormally activated, producing antibodies that target the body's own tissues, and causing various symptoms such as fatigue, joint pain, rash, and kidney damage. Recent reports suggest that TLR9 may be involved in the pathogenesis of SLE (JCI Insight. 2018 Mar 8;3(5):e96795). Under certain conditions, TLR9 recognizes self-DNA and activates TLR9 on B cells, leading to the production of autoantibodies such as anti-DNA. The excessive production of autoantibodies triggers an inflammatory cascade and a large-scale immune response, ultimately resulting in organ damage in the patient. In addition to secreting autoantibodies, B cells secrete cytokines that promote inflammation, activate specific T cells, and are involved in attacking and damaging normal tissues in the body (Respir Res, 2007, 8(1):72-80) (Immunol Res. 2012 Sep; 53(1-3): 58-77). Further studies have shown that the TLR9 signaling pathway is closely related to the development of SLE, with TLR9 levels significantly higher in SLE patients than in healthy individuals and closely associated with SLE activity (Exp Ther Med. 2019 Apr; 17(4): 3247-3254). This discovery spurred the development of TLR9-specific inhibitors, most of which inhibit the intracellular MyD88 signaling pathway but have not shown therapeutic effects in relevant animal models. Because B cells play a crucial role in the pathogenesis of SLE, research and development of a range of B cell-targeting drugs is attracting attention, including B cell depletion therapies targeting B cell surface markers (such as anti-CD20 antibodies), inhibition of B cell activators (such as anti-BAFF antibodies), and CAR-T therapies targeting B cells (such as anti-CD19CAR-T).However, while these treatments can effectively remove circulating B cells, they do not show sufficient inhibitory effects on B cells in lymphatic lesions (enlarged lymph nodes), resulting in extremely limited clinical benefits. At the same time, the removal of B lymphocytes or significant inhibition of their function can lead to side effects such as infections caused by humoral immunodeficiency. Therefore, for SLE, it is essential to develop drugs that can effectively inhibit pathologically activated B lymphocytes in circulation and in lymphatic organs simultaneously, while also maintaining the humoral immune function of B lymphocytes. B lymphocytes have unique in vivo circulatory pathways and homing characteristics (shown in Figure 1). If B lymphocytes can carry a special drug that targets specific B lymphocytes and migrate to the site of inflammation along with circulating in the body, the goal of "target recognition and inhibition" can be achieved, not only effectively inhibiting pathologically activated B lymphocytes but also reducing the decrease in humoral immunity and off-target side effects caused by B cell deficiency (shown in Figure 2).

[0004] Experiments by Jamin C et al. showed that when TLR9 expressed on the B cell membrane is simply activated without endocytosis and does not enter the cytoplasm, negative regulation of the TLR9 / MyD88 / NF-κB signaling pathway occurs (J Autoimmun. 2014 Jun;51:23-9.) (shown in Figure 3).

[0005] TLR9 can recognize palindromic sequences with unmethylated cytosine-phosphate-guanine oligonucleotides (CpG oligonucleotides, CpGODNs) as their core. CpG ODNs, TLR9 receptor agonists, can activate B cells by binding to the TLR9 receptor, inducing the production of various cytokines such as IL-6 and IFN-γ. B cells in the transitional and marginal zones of lymphoid follicles are highly sensitive to TLR9 stimulation, inducing activation, proliferation, and immunoglobulin production, and are involved in humoral and cellular immunity. CpG ODNs are short, single-stranded DNA fragments synthesized to contain unmethylated CpG dinucleotide sequences with specific sequences. CpG ODNs have a partial or complete phosphorothioate (PS) backbone. CpG ODNs differ in structural characteristics and immunological effects depending on the type, and are generally classified into three classes: A, B, and C. Class A CpG ODNs have a palindromic sequence containing CpG dinucleotides as their core, with poly(G) at both ends, and a phosphodiester bond backbone that is partially thiolated. The higher-order structure is formed by the palindromic sequence and poly(G), and mainly activates plasmacytoid dendritic cells (pDCs) but has weak activity against B cells. Class A mainly induces the production of many type I interferons, while inducing relatively weak IL-6. Class B CpG ODNs are linear due to complete thiolation modification and show strong immunostimulatory activity against B cells but cannot activate plasmacytoid dendritic cells. Class C CpG-ODNs are also completely thiolated, form dimers via a palindromic sequence, and possess the activity of both Class A and B CpG-ODNs, activating both plasmacytoid dendritic cells and B cells. When CpG ODN binds to TLR9, it is taken up into the cytoplasm via endocytosis, binds to myeloid differentiation factor 88 (MyD88), activates the nuclear factor kappa B (NF-κB) signaling pathway, and initiates downstream cytokine gene transcription. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] JCI Insight.2018 Mar 8;3(5):e96795 [Non-Patent Document 2] Respir Res, 2007, 8(1):72-80 [Non-Patent Document 3] Immunol Res.2012 Sep;53(1-3):58-77 [Non-Patent Document 4] Exp Ther Med.2019 Apr;17(4):3247-3254 [Non-Patent Document 5] J Autoimmun. 2014 Jun;51:23-9 [Overview of the project] [Problems that the invention aims to solve]

[0007] The object of the present invention is to find a B lymphocyte-specific drug (e.g., a TLR9-specific binding molecule) that, based on the principle of specific recognition of TLR9 to CpGODN, covalently binds to B lymphocytes, is reinjected into the patient, and travels along the specific in vivo circulation pathway of B lymphocytes to reach the lesion site and lymphoid organs (enlarged lymph nodes in SLE patients), inhibits or eliminates pathologically activated B lymphocytes, and achieves the therapeutic objective of the disease. [Means for solving the problem]

[0008] To achieve the above objectives, the present invention has designed an oligodeoxynucleotide (also called "ODN" in this invention) of unmethylated cytosine-phosphate-guanine dinucleotide, in which two identical first oligodeoxynucleotides having the sequence represented by SEQ ID.NO.1 are linked in tandem, or a first oligodeoxynucleotide and a second oligodeoxynucleotide having the sequence represented by SEQ ID.NO.2 are linked in tandem, based on the characteristics of the TLR9 recognition sequence. Here, the first oligodeoxynucleotide represented by SEQ ID.NO.1 is a B-class active ODN (CpG-B ODN) named "ODN1826," which strongly activates B cells and TLR9-mediated NF-κB signaling and is classified as a B-class CpG ODN that is a ligand for mouse TLR9. It mainly acts on B lymphocytes to stimulate the synthesis and secretion of IL-6, but its effect on the synthesis and secretion of INF-α is relatively weak.

[0009] In this invention, as a control of the above-mentioned ODN1826, a CpG-B ODN whose sequence is represented by SEQ ID.NO.3 and named "ODN 4084-F" was selected. ODN 4084-F is an inhibitory ODN belonging to the B class inhibitory ODN (Arthritis Res Ther. 2006, 8(1):203.) and can effectively inhibit TLR9-induced B lymphocyte activation (Arthritis Res Ther. 2009, 11(3):R79.), but the mechanism is unknown.

[0010] In one preferred embodiment, the two tandem-linked oligodeoxynucleotides described above are linked by two nucleosides, namely AT and TA, or by two other nucleosides, namely CG and GC, or directly linked.

[0011] In one more preferred embodiment, the above oligodeoxynucleotide has a sequence represented by SEQ ID.NO.4 and is named ODNACT-1 in this invention. The above sequence is composed of two ODN1826s represented by SEQ ID.NO.1 linked in tandem and joined together by AT. Alternatively, the above oligodeoxynucleotide has a sequence represented by SEQ ID.NO.5 and is named ODNACT-2 in this invention. The above sequence is composed of two ODN1826s represented by SEQ ID.NO.1 linked in tandem and joined together by CG. Alternatively, the above oligodeoxynucleotide has a sequence represented by SEQ ID.NO.6 and is named ODNACT-3 in this invention, and the above sequence is composed of an ODN1826 represented by SEQ ID.NO.1 and an A-class ODN represented by SEQ ID.NO.2 directly linked in tandem. The above oligodeoxyribonucleotides are labeled with dibenzocyclooctin (DBCO) and biotin at their 5' and 3' ends, respectively.

[0012] In the embodiments described above, more preferably, the 3' or 5' end of the oligodeoxynucleotide contains guanosine diphosphate fucose. The present invention aims to bind the above oligodeoxynucleotide to N-GlcNac of a cell membrane surface glycoprotein by enzymatic reaction with α-1,3-fucosyltransferase to form a stable glycosidic bond. For this purpose, the above technical means ensure that the oligodeoxynucleotide is supported so as to be provided for the enzymatic reaction with the α-1,3-fucosyltransferase donor substrate.

[0013] Next, the present invention provides cells to which the above-mentioned oligodeoxynucleotides are bound, wherein the cell membrane has an N-glycan branched chain containing N-acetylglucosamine and / or N-acetyllactosamine. The present invention uses α-1,3-fucosyltransferase to transfer guanosine diphosphate fucose contained in the 3' or 5' end of the above-mentioned oligodeoxynucleotide to GlcNAc (N-acetylglucosamine) and LacNac (N-acetyllactosamine) on the N-glycan branched chain by enzymatic reaction. Furthermore, as long as the surface glycoprotein N-GlcNac is present on the cell membrane, the above-mentioned oligodeoxynucleotides bind to N-GlcNac and stable glycosides so that the above-mentioned cells bind to the above-mentioned oligodeoxynucleotides, and the above-mentioned cells produce various biological effects of the oligodeoxynucleotides.

[0014] In one preferred embodiment, the cells are B lymphocytes.

[0015] Thirdly, the present invention provides the above-mentioned uses of cells in the preparation of pharmaceuticals for the treatment of tumors, autoimmune diseases, inflammatory diseases, and metabolic diseases.

[0016] In the treatment of tumor diseases, attaching one or more tumor-specific recognition antigens to immune cells, for example, attaching one or more tumor-specific recognition antigens such as anti-TROP2, CLDN18.2, EFGR, or VEGFR to a general-purpose CAR-T cell, can enhance the targeting ability of CAR-T cells, reduce off-target side effects, and improve antitumor effects.

[0017] An autoimmune disease refers to a disease state induced by the immune system of a living body mounting an immune response against self-components. As a result of activating an immune response against foreign antigens, the living body generally eliminates the antigens. However, when an immune response is activated against self-cells or tissue antigens, the self-cells or tissue are not easily and completely eliminated by the effector cells of the immune system and are continuously attacked, resulting in the living body reaching a disease state. TLR9 mediates innate and acquired immunity by recognizing pathogen-associated CpG ODN and is involved in the occurrence of various physiological and pathological processes such as inflammation, immunity, infection, and allergy. TLR9 / MyD88 / NF-κB is one of the important defense pathways of the living body against diseases. However, when the immune response becomes overly strong, it may cause damage to the living body (such as cell and tissue damage and dysfunction), leading to the occurrence or progression of autoimmune diseases. Currently, the induction mechanism of autoimmune tissue damage is considered to be mainly antibody-mediated autoimmunity and immune disorders caused by lymphocytes. It is suggested that B lymphocytes are abnormally activated, producing antibodies targeting self-tissues and further causing various symptoms such as fatigue, joint pain, rash, and kidney damage.

[0018] Therefore, in the case of B lymphocytes bound to the above-mentioned oligodeoxynucleotide according to the present invention, the oligodeoxynucleotide bound to the B lymphocyte can specifically bind to TLR9 of surrounding cells, but because it is covalently bound to the cell membrane surface, it cannot be endocytized, and therefore cannot activate the intracellular MyD88 / NF-κB signaling pathway. Furthermore, it can also inhibit the activated MyD88 / NF-κB signaling pathway through negative regulation. The bound cells may be cells in the blood circulation, such as red blood cells and B lymphocytes. This invention utilizes the unique in vivo circulation pathways and homing characteristics of B lymphocytes to bind ODN to the B lymphocyte membrane, thereby altering the pharmacokinetic properties of the drug, such as distribution, metabolism, and excretion, maximizing the therapeutic effects of the drug and reducing side effects. Furthermore, by binding various therapeutic agents, including antibodies, polypeptides, small molecules, and nucleic acids, to the cell membranes of B cells, stem cells, and progenitor cells using the method according to this invention, it is possible not only to adequately maintain the functions of various cells but also to exert the properties of the bound drug, causing it to migrate to inflammatory sites where pathologically activated B cells are concentrated, thereby achieving the objective of "target recognition," effectively inhibiting pathologically activated B lymphocytes, reducing off-target side effects, and realizing targeted therapy. For example, by binding an immune checkpoint activating antibody or a fusion protein such as PD-L1 recombinant protein to the surface of B cells, it is possible to specifically recognize and inhibit autoantigen-activated T lymphocytes within the germinal center of lymph nodes by utilizing the antigen-presenting action of B cells and the T lymphocyte activity inhibitory action of PD-L1, thereby improving the therapeutic effect of autoimmune diseases. Therefore, the B lymphocytes described above according to the present invention can be used in the preparation of pharmaceuticals for treating autoimmune diseases.

[0019] In one preferred embodiment, the above-mentioned autoimmune diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, acute glomerulonephritis, rheumatic heart disease, and diabetes mellitus.

[0020] Regarding inflammatory diseases, they usually include autoinflammatory diseases (such as the above-mentioned SLE) and inflammatory diseases caused by other pathogenic microorganism infections such as inflammation of joints (arthritis) and the heart (carditis) due to streptococcal infection in the pharynx. Good prognosis can be obtained by using antibacterial therapy with high sensitivity in the acute phase. For chronic diseases that develop due to insufficient acute-phase treatment, the treatment strategy is the same as that for autoimmune diseases, mainly aiming to suppress the immune function of T cells, especially T cells around the lesion. Fusion proteins of immune checkpoints such as recombinant proteins like PD-L1 and CTLA-4 bind to B lymphocytes and are presented to T cells to suppress their functions.

[0021] Regarding metabolic diseases, by binding a metabolic-related enzyme such as uricase to the surface of blood cells, blood uric acid can be removed and the circulating uric acid level can be decreased. At the same time, B cells transport uricase to tissues, remove uric acid crystals deposited in tissues to relieve pain, and achieve the goal of treating refractory gout and acute hyperuricemic nephropathy.

[0022] Finally, the present invention (1) a step of modifying the 3'-end of the above oligodeoxynucleotide with DBCO and modifying the 5'-end with biotin; (2) adding an excessive amount of guanosine diphosphate fucose-azide (GDP-Azido-Fucose, purchased from R&D Systems, product number ES101-100) to the solution of the modified oligodeoxynucleotide obtained in step (1) and incubating to conjugate the two to obtain GDP fucose-oligodeoxynucleotide; (3) A method for conjugating oligodeoxynucleotides to B lymphocytes is provided, comprising the step of adding the GDP fucose-oligodeoxynucleotide obtained in step (2) and α-1,3-fucosyltransferase obtained in step (2) to a B lymphocyte suspension and incubating to obtain B lymphocytes to which the oligodeoxynucleotides are bound. In the incubation process described above, the GDP fucose-oligodeoxynucleotides form glycosidic bonds with the GlcNac receptor molecule of the target cell membrane surface glycoprotein, and ODN-labeled B lymphocytes are obtained.

[0023] In one preferred embodiment, in step (3), 5 × 10 B cells 8 Add 100 μg / mL of α-1,3-fucosyltransferase and 10 μM of GDP-fucose-oligodeoxynucleotide prepared in step (2) to each of the mL values. [Effects of the Invention]

[0024] The unmethylated cytosine-phosphate-guanine dinucleotide oligodeoxynucleotide according to the present invention does not affect the function of the B lymphocytes themselves even when bound to the surface of B lymphocytes. Therefore, it ensures the unique in vivo circulation and homing characteristics of B lymphocytes, and along with in vivo circulation, it can target migration to diseased lymphoid organs such as lymph nodes, achieving the goal of "target recognition and inhibition." This not only effectively inhibits pathologically activated B lymphocytes but also reduces the decrease in humoral immunity and off-target side effects caused by B cell deficiency, thereby improving therapeutic efficacy. [Brief explanation of the drawing]

[0025] [Figure 1] This is a schematic diagram of the circulatory pathway of B lymphocytes in the body. [Figure 2] This is a schematic diagram illustrating the treatment of local or systemic lymphadenopathy of SLE with ODN-labeled B lymphocytes. [Figure 3] This schematic diagram illustrates the opposing functions of TLR9 on the cell membrane surface and on the endosomal / lysosomal membrane in the MyD88 / NF-κB signaling pathway. [Figure 4] TLR9-activated ODN1826 can stimulate the activation of mouse B cells and improve the secretion levels of mIL-6 and mTNF-α. [Figure 5] ODN-ACT1, ODN-ACT2, and ODN-ACT3 can significantly increase the secretion levels of mIL-6 and mINF-α under identical conditions. [Figure 6] ODN 4084-F can inhibit the activation of naive B cells by ODN1826. [Figure 7] ODN 4084-F cannot inhibit activated B cells. [Figure 8] ODN1826 is labeled on the cell membrane surface. [Figure 9] The labeling effect of ODN1826 on the cell membrane surface is not affected by the position of biotin or by PS and PO binding. [Figure 10] B cells labeled with ODN1826 still retain B cell function. [Figure 11] B cells labeled with ODN1826 cannot activate each other. [Figure 12] This is a schematic diagram of an experiment demonstrating the inhibitory effect of ODNACT-2 on activated B cells by B cells. [Figure 13] B cells labeled with ODNACT-2 can effectively inhibit activated B cells. [Modes for carrying out the invention]

[0026] The present invention will be described in more detail below based on specific embodiments, and its advantages and features will become even clearer from the following description. However, these embodiments are merely illustrative and do not limit the scope of the claims of the present invention in any way.

[0027] Example 1. Activation of mouse B cells by the activated form of TLR9, ODN1826.

[0028] The oligonucleotides in Table 1 were synthesized by Suzhou Hong Xun Biotechnology Co., Ltd. based on sequences SEQ ID NO.1 to SEQ ID NO.6. Here, SEQ ID NO.1 is based on ODN1826, and to facilitate measurement, the 5' and 3' ends were labeled with dibenzocyclooctin (DBCO) and biotin, respectively. To prevent cleavage and degradation by nucleases, the phosphodiester bonds between the nucleotides at both ends of the ODN1826-1 and ODN1826-2 sequences were modified with phosphorothioate bonds. The difference between ODN1826-1 and ODN1826-2 is that the modification positions of DBCO and biotin are reversed. In ODN1826-3, the modification positions of DBCO and biotin are the same as in ODN1826-2, but all nucleotides in ODN1826-3 are linked by phosphodiester bonds.

[0029] [Table 1]

[0030] The testing procedure for ODN-activated mouse B lymphocytes is as follows:

[0031] 1. Preparation of B cell suspension

[0032] Mouse B lymphocytes were collected from the fresh spleens of (C57BL / 6J) mice, lightly ground on a ground glass sheet, resuspended in PBS (Solarbio, P1020), and collected in a 15 mL centrifuge tube. After removing PBS by centrifugation at 500 g for 5 minutes, each spleen was resuspended in 5 mL of RBC Lysis Buffer (Solarbio, R1010), and the red blood cells were lysed at room temperature for 5 minutes. After lysis, 10 mL of PBS was added to stop the reaction, and after thorough mixing, the erythrocyte lysate was removed by centrifugation at 500 g for 5 minutes. The cells were washed with PBS 10 mL each for a total of three times. After the third resuspendion, the cell suspension was filtered through a 70 μm mesh (NEST, 258368) to remove large spleen tissue. A small amount of cell suspension was then collected and the cell count was counted (Dimai Bio-Technology, model DF55 Vet). After centrifugation, the cells were resuspended in RPMI1640 medium (Gibco, 61870-036) + 10% FBS (Sigma, F8687-100ML) to obtain a single spleen cell suspension, and the concentration was reduced to 10. 6 The solution was adjusted to 10 / mL and used for subsequent experiments. To obtain a higher purity of B cells, the single spleen cell suspension was prepared as in the previous step. 6 A solution of 20 μg / mL was prepared, and 10 mL of it was added to a 10 cm culture dish (Corning, 430167). A final concentration of 20 μg / mL LPS (Solarbio, L8880) was added, and the cells were cultured at 37°C and 5% CO2 for 48 hours. After culturing, a small amount of cells were collected, stained with CD19-FITC (Elabscience, E-AB-F0986UC, 1:500 dilution), and measured by flow cytometry (Beijing Challen Biotechnology Co., Ltd., model MateCyte). If the CD19 positivity rate was 95% or higher, the B cell preparation was considered successful. The B cells were then cultured in 1640 complete medium at a concentration of 10 6 The solution was adjusted to / mL and used in subsequent experiments.

[0033] 2. Activation of mouse B cells by ODN1826

[0034] The single cells prepared using the above procedure were seeded in a 96-well plate (Corning, 3799) by adding 200 μL of cell suspension to each well. Corresponding concentrations of ODN1826-1, 2, and 3 (Suzhou Junqi, S9622101703026) were added, and the cells were cultured at 37°C and 5% CO2 for 24 hours. After culturing, the supernatant was collected by centrifugation, and mRNA (Solarbio, SEKM-0007) and mTNF-α (Solarbio, SEKM-0034) were measured by ELISA. The measurements were performed according to the kit instructions, and absorbance was read at two wavelengths, OD450 nm and OD630 nm, using a microplate reader (Molecular Devices, model number spetramax paradigm).

[0035] Figure 4 shows that the addition of ODN1826 stimulated the secretion of IL-6 (Figure 4A) and TNF-α (Figure 4B) from splenic cells compared to a control group without ODN1826, and that the secretion of IL-6 and TNF-α increased with increasing ODN1826 concentration.

[0036] As shown in Figure 4, all three oligonucleotides are based on ODN1826 and belong to the B-class TLR9 agonist group. Despite slight differences in the modifications of the three oligonucleotides, no significant difference was observed in B lymphocyte activation, and the secretion of IL-6 and TNF-α increased with increasing oligonucleotide concentration. Therefore, all subsequent TLR9-activating oligonucleotides were labeled with DBCO at the 5' end and biotin at the 3' end, and phosphodiester bonds were used for nucleotide linkages.

[0037] Example 2. Activation of mouse B cells by ODN-ACT1, ODN-ACT2, and ODN-ACT3

[0038] As a TLR9 agonist, ODN1826 did not exhibit ideal activation in Example 1, and even doubling its concentration only resulted in a 30% increase in secreted mRNA. Therefore, it was hypothesized that activation could be enhanced by tandem linking of ODN1826. SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 are named ODNACT-1, ODNACT-2, and ODNACT-3, respectively. ODN ACT-1 linked two B-class ODNs via AT (adenine-thymine), ODN ACT-2 linked two B-class ODNs via CG (cytosine-guanine), and ODN ACT-3 directly linked a B-class ODN to an A-class ODN.

[0039] The mouse B lymphocytes were derived from high-purity B lymphocytes amplified in vitro. The test procedure was the same as in Example 1.

[0040] As shown in Figure 5, ODN ACT-1, ODN ACT-2, and ODNACT-3 all showed stronger B cell activation effects than ODN1862-3 under the same concentration and number of B cells. In Figure 5, A represents IL-6 measurement, B represents INF-α measurement, and C represents IgG measurement. The three TLR9 activators, ODN ACT-1, ODN ACT-2, and ODNACT-3, showed different effects on B cell activation. ODN ACT-2, as a B-class activator, showed stronger stimulation of IL-6 secretion and was the most active of the four ODN activators. This result is consistent with the expectation that ODN ACT-2, although both ODN ACT-1 and ODN ACT-2 consist of two B-class ODNs linked in tandem, uses CpG at the binding site and has a stronger binding affinity to TLR9. Surprisingly, ODN ACT-2 also showed the strongest INF-α activation effect. ODNACT-3 tandemizes one B-class ODN and one A-class ODN. While literature shows that A-class ODNs have a more significant IFN-α activation effect than B-class ODNs, studies have reported that ODN ACT-2 still shows a stronger IFN-α secretion stimulating effect than ODNACT-3. In summary, ODN ACT-2 showed a more pronounced stimulating effect on B cells.

[0041] Example 3. Experiment on B lymphocyte activation using ODN 4084-F

[0042] ODN 4084-F is a novel inhibitory oligonucleotide (ODN) consisting of two nucleotide triplets, one proximal CCT and one distal GGG, separated by four nucleotides. ODN 4084-F is the shortest inhibitory ODN with the sequence represented by SEQ ID NO. 3, and as a B-class inhibitory ODN, it can effectively inhibit TLR9-mediated B cell activation (Arthritis Res Ther. 2009, 11(3):R79). Literature reports indicate that the inhibitory effect of ODN 4084-F is dependent on phosphorothioate bonds, and we confirmed this phenomenon. When synthesizing the ODN 4084-F oligonucleotide, we selected phosphorothioate bonds instead of phosphodiester bonds.

[0043] As shown in Figure 6, ODN 4084-F failed to stimulate mRNA production in mouse B lymphocytes, while ODN1826 effectively activated B lymphocytes. When B lymphocytes were co-cultured after mixing ODN 4084-F and ODN1826 in a fixed ratio, ODN1826-mediated mRNA production gradually decreased as the concentration of ODN 4084-F gradually increased, which is consistent with previously reported literature.

[0044] As shown in Figure 7, when B lymphocytes are initially activated by LPS and / or mRNA (similar to pathologically activated B lymphocytes in SLE), activated B cells can still be further activated by ODN1826, but the addition of ODN 4084-F could not inhibit mRNA production. Therefore, ODN 4084-F can prevent the activation of naive B cells by ODN1826, but it did not show a significant inhibitory effect on already activated B cells. Consequently, ODN 4084-F is not suitable as a therapeutic agent, especially for active SLE, which is likely one reason why ODN 4084-F is not clinically used as a TLR9 inhibitor.

[0045] The test procedure is as follows:

[0046] B cells 10 6 Prepare a cell suspension of 1 / mL according to the method described in Example 1, add 200 μL of cell suspension to each well, and seed in a 96-well plate (Corning, 3799). Add 1 μM ODN1826-3 (Suzhou Junqi, S9622101703026) and / or 1 μM, 3 μM, or 10 μM ODN4084-F (Suzhou Hong Xun Biotechnology Co., Ltd., CN32260) and culture at 37°C and 5% CO2 for 24 hours. After culturing, the supernatant was collected by centrifugation and mRNA (Solarbio, SEKM-0007) was measured by ELISA.

[0047] To obtain activated B cells, prepared B cells were stimulated with LPS (Solarbio, L8880) at a final concentration of 20 μg / mL and mMil-4 (Genscript, Z02996) at a final concentration of 2.5 ng / mL for 24 hours at 37°C and 5% CO2. The activated B cells were seeded into 96-well plates (Corning, 3799) by adding 200 μL of cell suspension to each well, and cultured for 24 hours at 37°C and 5% CO2 with ODN1826-3 (Suzhou Junqi, S9622101703026) at a final concentration of 1 μM and ODN4084-F (Suzhou Hong Xun Biotechnology Co., Ltd., CN32260) at a final concentration of 1 μM and 1 μM or 10 μM. After culturing, the supernatant was collected by centrifugation and mMil-6 (Solarbio, SEKM-0007) was measured by ELISA.

[0048] Example 4. Labeling of oligonucleotide chains to B lymphocytes

[0049] Helicobacter pylori-derived α-1,3-fucosyltransferase (FucT) can transfer fucose from guanosine diphosphate fucose (GDP-Fucose) derivatives to GlcNAc (N-acetylglucosamine) and LacNac (N-acetyllactosamine) in N-glycan branched chains. This property allows ODN containing GDP-fucose at its 3' or 5' end to be linked to N-GlcNac, a cell membrane surface glycoprotein, by the action of FucT, forming a stable glycosidic bond. To achieve this objective, the 3' and 5' ends of the oligonucleotide chain were first modified with DBCO and biotin, respectively, during synthesis (see Table 1). ODN was mixed with an excess amount of guanosine diphosphate fucose-azide (GDP-Azido-Fucose) (purchased from R&D Systems, catalog number ES101-100) and incubated at room temperature for 30 minutes. Then, B lymphocyte suspension was added, along with FucT, and incubated for another 30 minutes. After washing the cells three times, the ODN on the cell membrane surface was measured using PE-labeled streptavidin (Streptavidin-PE, SA-PE), which is shown in Figure 8. The specific procedure is as follows:

[0050] 1. The preparation of α-1,3-fucosyltransferase mutants derived from Helicobacter pylori is as follows:

[0051] (1) Suzhou Junqi Biotechnology Co., Ltd. was requested to synthesize plasmid DNA expressing α-1,3-fucosyltransferase (strain ATCC 700392 / 26695) derived from Helicobacter pylori, and vector pET41a (MilliporeSigma TM The plasmid was cloned into pET-41a(+) DNA Vector (catalog number: 70-556-3). 2 μL of the plasmid was added to 100 μL of BL21(DE3) competent cells (ThermoFisher, catalog number: EC0114), immediately mixed homogeneously, and allowed to stand on ice for 30 minutes.

[0052] (2) The patient underwent a heat shock at 42°C for 90 seconds, and was immediately cooled in an ice bath for 2 minutes.

[0053] (3) Add 500 uL of LB medium and culture with shaking at 37°C and rpm <= 200 for 60 minutes.

[0054] (4) Centrifuge at 6000 rpm for 1 minute, discard most of the supernatant, leaving approximately 100-150 μL, resuspend the bacterial cells, spread them onto an Amp-containing LB plate, and incubate overnight at 37°C.

[0055] (5) Low-level expression: A single colony was inoculated into 1 mL of Amp-resistant LB medium and cultured with shaking at 37°C and 220 rpm for approximately 5 hours. Then, 2.5 mL of Amp-containing LB liquid medium was added to the centrifuge tube used in the previous step and cultured with shaking overnight at 37°C and 220 rpm.

[0056] (6) The bacterial suspension cultured overnight was transferred to 20 mL of Amp-containing LB medium in a 1:50 ratio and cultured at 37°C and 220 rpm until the OD600 reached 0.6 (approximately 3 hours). Then, 0.5 mM IPTG was added, and the culture was incubated at 30°C and 220 rpm for 6 hours.

[0057] (7) The OD600 of the culture medium was measured, 10 OD of bacterial suspension was collected, centrifuged at 10,000 rpm for 2 minutes, and the supernatant was removed.

[0058] (8) The bacterial cells were resuspended in 1 mL of dissolving solution (10 mM Tris-HCl, pH 8.0), and the cells were sonicated on ice under the following conditions: 130 W, 4 minutes, 3 seconds on / 3 seconds off.

[0059] (9) After the ultrasound was completed, the lysis solution was centrifuged at 12,000 rpm for 10 minutes to obtain the supernatant, which was then ultracentrifuged at 4°C and 125,000 g.

[0060] (10) The supernatant was applied to a HiTrap chelate HP column according to the manual and eluted with 20 mM imidazole solution. The eluted fraction was pooled, the solvent was replaced with 50 mM Tris buffer (pH 8.0) by dialysis, and then further purified by gel filtration chromatography (superdex200, GE Healthcare) to obtain a high-purity protein with a homogeneity of 98% or more.

[0061] (11) Protein concentrations were measured using the Bio-Rad protein assay kit based on the Bradford method with bovine serum albumin as the standard substance.

[0062] 2. Preparation of GDP-Fucose-ODN

[0063] GDP-azido-fucose (purchased from R&D Systems, catalog number ES101-100) 100 μM and ODN 100 μM were uniformly mixed in a 1:2 ratio and incubated at room temperature for 30 minutes to obtain GDP-fucose-ODN with a theoretical concentration of 33 μM.

[0064] 3. Binding of GDP-fucose-ODN to the cell membrane.

[0065] After washing the B cells with PBS, the culture medium was thoroughly removed before use. 5 × 10⁶ B cells 8 To the PBS solution, 100 μg / mL of FucT and 10 μM of GDP-Fucose-ODN prepared in the previous step were added, and the mixture was incubated at room temperature for 30 minutes. The mixture was washed with PBS, centrifuged at 500 g for 5 minutes, and the supernatant was removed. The B cells were resuspended in PBS, stained with streptavidin-PE (BioLegend, Inc., product no. 405204, 1:200 dilution), co-incubated at 4°C for 30 minutes, washed once with PBS, measured by flow cytometry (Beijing Challen Biotechnology Co., Ltd., product no. MateCyte), and analyzed with NovoExpress software.

[0066] As shown in Figure 8, ODN could be rapidly labeled onto the surface of B lymphocytes at room temperature. To compare the effects of DBCO and biotin positions, and PS and PO binding on the labeling effect, B lymphocytes were labeled with ODN1826-1, ODN1826-2, and ODN1826-3 under the same conditions. As shown in Figure 9, all three ODN1826-1, ODN1826-2, and ODN1826-3 effectively labeled the surface of B lymphocytes, and the positions of DBCO and biotin, and PS and PO binding, did not significantly affect the labeling of mouse B lymphocytes.

[0067] Example 5. Verification experiment of B cell function preservation in oligonucleotide-labeled B cells.

[0068] Lymphocyte homing is a characteristic of B lymphocytes, referring to directional migration that includes migration from peripheral tissues to central lymphoid organs, migration from central lymphoid organs to peripheral lymphoid organs, lymphocyte recirculation, and the migration of lymphocytes to inflammatory sites (such as the skin, intestinal mucosa, and synovial joints). The essence of the homing process is the interaction between receptors on the lymphocyte membrane surface and adhesion molecules on vascular endothelial cells in each tissue and organ. During the preparation of ODN-labeled lymphocytes, B lymphocytes are not subjected to chemical or physical treatment, and the amino acids on the cell membrane surface are not affected. To confirm that ODN-labeled B lymphocytes still retain complete membrane function, the reactivity of B lymphocytes to LPS, IL-4, and TLR9 activators was compared before and after ODN labeling. As shown in Figure 10, ODN-labeled B lymphocytes showed a good response to TLR9 activators, similar to before labeling, and exhibited dose-dependent behavior. Therefore, the labeling method according to the present invention is non-invasive to B lymphocytes, and the process is mild. This method allows B lymphocytes carrying ODN to retain their characteristics when reinjected into the body, enabling them to localize to diseased B lymphocyte tissue and exert a therapeutic effect by utilizing their unique homing and migration pathways.

[0069] 1. The test procedure in Figure 10 is as follows:

[0070] (1) B cells at 10 6 / mL were prepared according to the method described in Example 1 and used.

[0071] (2) B cells bound with GDP-Focuse-ODN were prepared according to the method described in Example 4. After successful detection by flow cytometry, the cells were resuspended to a concentration of 1E6 / mL.

[0072] (3) 200 μL of the prepared cell suspension was added to each well, and the cells were seeded in a 96-well plate (Corning, 3799). ODN1826-3 (Suzhou Junji, S9622101703026) with final concentrations of 0.25 μM, 0.5 μM, and 1 μM was added, and the cells were cultured at 37 °C and 5% CO2 for 24 hours. After the culture, the supernatant was collected by centrifugation, and mIL-6 (Solarbio, SEKM-0007) was measured by ELISA.

[0073] When culturing B cells labeled with ODN1826, to answer whether cells can recognize TLR9 on the cell membrane of the other cells through ODN1826 on their respective cell membranes and cause mutual activation, B lymphocytes were extracted from three mice each. For a part of them, ODN1826 was labeled on the cell membrane surface according to the method of Example 4. Naïve B lymphocytes and ODN-labeled B lymphocytes were cultured for 24 hours each, the culture supernatant was collected, and the level of mIL-6 was measured. As a positive control signal for B lymphocyte activation, a part of the naïve B lymphocytes was added to free ODN1826 and cultured. As a result, as shown in Figure 11, even when culturing B cells labeled with ODN1826, the level of mIL-6 in the culture supernatant did not increase. It was suggested that even if ODN1826 is labeled on the cell membrane, cells do not mutually activate each other and maintain the same state as naïve B cells.

[0074] 2. The test procedure in Figure 11 is as follows.

[0075] (1) Fresh spleens were collected from three mice, and B cells at 106 Each solution was prepared in a / mL concentration and used.

[0076] (2) In addition, B cells bound to GDP-fucose-ODN were prepared according to the method described in Example 4, and after successful detection by flow cytometry, 10 6 Resuspend until the concentration reaches / mL.

[0077] (3) The prepared cells described above were seeded in a 96-well plate (Corning, 3799) by adding 200 μL of cell suspension to each well. A portion of the naive B lymphocytes (the rightmost column of each mouse in the figure) were then treated with ODN1826-3 (Suzhou Junqi, S9622101703026) at a final concentration of 1 μM, and the cells were cultured at 37°C and 5% CO2 for 24 hours. After the culture was complete, the supernatant was collected by centrifugation, and mRNA (Solarbio, SEKM-0007) was measured by ELISA.

[0078] Example 6. Use of ODNACT-2 on activated B cells

[0079] As described in Example 5, B cells labeled with ODN1826 still possessed the characteristics of naive B cells, and the cells were not mutually activated by ODN. Therefore, if ODN1826 labeled on the cell membrane binds to and activates TLR9 without inducing TLR9 / ODN endocytosis, it negatively regulates MyD88, demonstrating the feasibility of a drug as an immunosuppressant. ODNACT-2 showed stronger binding ability to TLR9 than ODN1826. To confirm the inhibitory effect of ODNACT-2-labeled B cells on activated B lymphocytes, B cells from the same mouse were divided into groups as shown in Figure 12: Group A was stimulated with LPS and mIL-4 to simulate activated B lymphocytes from SLE patients, Group B was bound and labeled with ODNACT-2, and Group C used naive B lymphocytes as a control. Cells from group A were cultured together with groups B and C, respectively, and after 24 hours, the level of mL-6 in the culture supernatant was measured (0.21 ng / mL ± 0.013 ng / mL for group A+C; 0.11 ng / mL ± 0.038 ng / mL for group A+B). As shown in Figure 13, ODNACT-2 labeled on the cell membrane surface was able to effectively inhibit activated B cells.

[0080] The test procedure is as follows:

[0081] 1. Activated B cells (i.e., group A in the figure) were obtained according to the method of Example 3, 100 μL of cell suspension was added to each well, and the cells were seeded in a 96-well plate (Corning, 3799).

[0082] 2. B cells 10 according to the method described in Example 1 6 A solution of / mL was prepared (i.e., group C in the figure).

[0083] 3. Following the method described in Example 4, B cells bound to GDP-fucose-ODN were prepared and successfully detected by flow cytometry. 6 The solution was resuspended until it reached a concentration of / mL (i.e., group B in the figure).

[0084] 4. Prepared cells from groups C and B were added to each well by adding 100 μL of cells to the wells containing seeded cells from group A, and then cultured at 37°C and 5% CO2 for 24 hours. After culturing, the supernatant was collected by centrifugation, and mMil-6 (Solarbio, SEKM-0007) was measured by ELISA. [Industrial applicability]

[0085] As described above, the present invention provides a method for designing and synthesizing highly active ODN and for binding it to the surface of B lymphocytes. According to the method of the present invention, it is possible to immediately bind OND to the surface of B lymphocyte membranes under mild conditions without affecting the original function of the cells. Therefore, it is possible to ensure the in vivo circulation and homing characteristics unique to B lymphocytes, enable targeted migration to diseased lymphoid organs such as lymph nodes, inhibit B lymphocytes activated under pathological conditions, achieve therapeutic objectives, enhance tissue targeting, and improve therapeutic effects.

Claims

1. An oligodeoxynucleotide of a non-methylated cytosine-phosphate-guanine dinucleotide, characterized in that the oligodeoxynucleotide is composed of two identical first oligodeoxynucleotides having the sequence represented by SEQ ID. NO. 1 linked in tandem, or a first oligodeoxynucleotide and a second oligodeoxynucleotide having the sequence represented by SEQ ID. NO. 2 linked in tandem.

2. The oligodeoxynucleotide of a non-methylated cytosine-phosphate-guanine dinucleotide according to claim 1, characterized in that the tandem-linked oligodeoxynucleotide is linked in tandem by one adenine and one thymine, or by one cytosine and one guanine, or directly in tandem.

3. The oligodeoxynucleotide of a non-methylated cytosine-phosphate-guanine dinucleotide according to claim 2, characterized in that the sequence of the oligodeoxynucleotide is represented by SEQ ID. NO. 4, SEQ ID. NO. 5, or SEQ ID. NO.

6.

4. The oligodeoxynucleotide of a non-methylated cytosine-phosphate-guanine dinucleotide according to any one of claims 1 to 3, characterized in that guanosine diphosphate fucose is included at the 3' or 5' end of the oligodeoxynucleotide.

5. A cell to which an oligodeoxynucleotide of a nonmethylated cytosine-phosphate-guanine dinucleotide described in claim 4 is bound, wherein the cell membrane of the cell has an N-glycan branched chain containing N-acetylglucosamine and / or N-acetyllactosamine.

6. The cell according to claim 5, characterized in that the cell is a B lymphocyte or a T lymphocyte.

7. Use of cells according to claim 6 in the preparation of a pharmaceutical product for treating tumors, autoimmune diseases, inflammatory diseases or metabolic diseases.

8. The use according to claim 7, characterized in that the aforementioned autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, acute glomerulonephritis, rheumatic heart disease, and diabetes.

9. A method for binding oligodeoxynucleotides according to any one of claims 1 to 3 to B lymphocytes, (1) A step of modifying the 3' end of an oligodeoxynucleotide according to any one of claims 1 to 3 with DBCO, and then modifying the 5' end with biotin, (2) Add an excess amount of guanosine diphosphate fucose-azide to the solution of the modified oligodeoxynucleotide obtained in step (1) and incubate to obtain GDP fucose-oligodeoxynucleotide, (3) A method characterized by comprising the step of adding the GDP fucose-oligodeoxynucleotide and α-1,3-fucosyltransferase obtained in step (2) to a B lymphocyte suspension and incubating to obtain B lymphocytes to which oligodeoxynucleotides are bound.

10. In step (3), B cells 5 × 10 ８ The method according to claim 9, characterized by adding 100 μg / mL of FucT and 10 μM of GDP fucose-oligodeoxynucleotide prepared in step (2) to each of the above.

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