A CpG oligonucleotide

Abstract

The present application discloses a CpG oligonucleotide, including a sequence encoding CpG TY001 or a sequence encoding CpG TY002. The CpG oligonucleotide includes modification of 3'OH or 5'OH groups, modification of nucleotide bases, modification of sugar components, backbone modification and modification of phosphate groups. Contains naturally occurring or modified non-natural bases, modified sugars, phosphates and / or ends. Contains one or more ribonucleotides, deoxyribonucleotides, modified sugars or sugar analogs. Has a homogeneous, heterogeneous or chimeric backbone. Contains modification of one or more phosphate groups. More than 50%, more than 60%, more than 70%, more than 80%, more than 90% of the nucleotides have modifications of phosphate groups.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine, and specifically relates to a CpG oligonucleotide. Background Art

[0002] CpG oligodeoxynucleotides are a new type of immunostimulant discovered in recent years. Their chemical nature is a deoxyoligonucleotide containing cytosine guanine dinucleotides. They have an immune response similar to that of natural CpG pattern recognition receptors and can bind to Toll-like receptors (TLRs) on the cell membrane. For example, patent application No. WO2001051083A3 discloses an immunostimulatory composition comprising saponin and CpG oligodeoxynucleotides, which records a CpG oligodeoxynucleotide (involving CpG1826 and CpG7909). For example, patent application No. 202411351154.5 discloses a new adjuvant recombinant herpes zoster vaccine composition and its preparation and application (involving CpG1018). However, due to the structural diversity of CpG oligodeoxynucleotides (the effects of CpG adjuvants of different sequences vary greatly).

[0003] The above-mentioned Toll-like receptors (TLRs) are transmembrane proteins that mainly act as sensors of microbial components, identifying bacterial and viral components. In mammalian infections and some non-infectious diseases, TLRs constitute a major defense mechanism. So far, 10 human (TLRs 1-10) and 12 mouse TLRs (TLRs 1-9 and TLRs 11-13) have been identified, among which TLR9 is expressed on human plasmacytoid dendritic cells (pDCs) and B cells, as well as various cells of the mouse myeloid lineage (including monocytes, macrophages and conventional dendritic cells).

[0004] CpG oligodeoxynucleotides bind to Toll-like receptors (TLRs) on the cell membrane and effectively trigger mammalian immune responses through the TLR9 signaling pathway. TLR9 specifically recognizes CpG motifs contained in bacteria, viruses, plasmids, or synthetic double-stranded or single-stranded oligonucleotides. CpGODNs are immunomodulatory synthetic oligonucleotides specifically designed to stimulate TLR9. So far, four types of synthetic CpG ODNs have been described, including K-type ODN (also known as B-type), D-type ODN (also known as A-type), C-type ODN, and P-type ODN, each with different structures and biological properties.

[0005] Currently, the only CpGODN that has been approved is CpG 1018, and there are relatively few types of vaccines containing this adjuvant. Only vaccines against hepatitis B and COVID-19 have been approved. Although the patent with patent application number 2020115461504 discloses a nucleic acid drug complex and the technology of its use, records a CpG oligonucleotide sequence that binds to the TLR9 receptor and is used to activate TLR9, and the complex is used as a cancer drug, the patent with patent application number 202280054322.7 discloses a sequential, innate and adaptive immune regulation for cancer treatment, and records a TLR9 agonist for treating cancer, and the patent with patent application number 201780067972.4 discloses the immunoregulatory effect of a TLR9 agonist on cancer treatment, which records a method for treating cancer patients, which includes administering an oligonucleotide TLR9 agonist to a cancer patient intratumorally, but currently no CpGODN has been approved for cancer treatment. Therefore, in view of the heavy burden of infectious diseases and cancer worldwide, there is still a need to develop TLR9 agonists with better activity to apply them to the prevention and treatment of infectious diseases and cancer. Summary of the invention

[0006] The purpose of the present invention is to solve the problems of current TLR9 agonists and provide a CpG oligonucleotide, including a CpG oligonucleotide, such as a sequence encoding CpG TY001 or a sequence encoding CpG TY002; the sequence of CpGTY001 is as follows: TCG CTT CGAAGC CAA CGT TGG; the sequence of CpG TY002 is as follows: TCG CAA CGT TGC CT CGAAGG.

[0007] As an improvement to the CpG oligonucleotide described in the present invention, the sequence includes modification of 3'OH or 5'OH groups, modification of nucleotide bases, modification of sugar components, modification of backbones and modification of phosphate groups.

[0008] As a further improvement to a CpG oligonucleotide of the present invention, the sequence contains naturally occurring or modified non-naturally occurring bases, modified sugars, phosphates and / or ends. In addition to phosphodiester bonds, phosphate modifications include, but are not limited to: methyl phosphonates, phosphorothioates, phosphoramidates (bridged or non-bridged), phosphotriesters and phosphorodithioates, and can be used in any combination. In certain embodiments, the CpG oligonucleotide has only thiophosphate bonds, only phosphodiester bonds, or a combination of phosphodiester bonds and thiophosphate bonds.

[0009] As a further improvement to a CpG oligonucleotide of the present invention, one or more ribonucleotides, deoxyribonucleotides, modified sugars or sugar analogs are included in the sequence. Therefore, in addition to ribose and deoxyribose, the sugar moiety can also be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose and sugar analog cyclopentyl. The sugar can be in the form of pyranosyl or furanosyl. In CpG oligonucleotides, the sugar moiety is preferably a furanoside of ribose, deoxyribose, arabinose or 2'-O-alkyl ribose, and the sugar can be connected to various heterocyclic bases in an end group isomerization configuration. Sugar modifications known in the art can also be made, such as 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA and 2'-alkoxy-or amino-RNA / DNA chimeras and others described herein, and combined with any phosphate modification. Examples of base modifications include, but are not limited to, the addition of electron-withdrawing moieties to C-5 and / or C-6 of cytosine (e.g., 5-bromocytosine, 5-chlorocytosine, 5-fluorocytosine, 5-iodocytosine) of CpG oligonucleotides and C-5 and / or C-6 of uracil (e.g., 5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil) of CpG oligonucleotides. The application of base modifications in the palindromic sequence of CpG oligonucleotides should not interfere with the self-complementarity of the bases involved in Watson-Crick base pairing. However, outside of the palindromic sequence, modified bases can be used without this restriction. For example, 2'-O-methyl-uridine and 2'-O-methyl-cytidine can be used outside of the palindromic sequence, however, 5-bromo 2'-deoxycytidine can be used inside and outside the palindromic sequence. Other modified nucleotides that can be employed both within and outside of palindromic sequences include 7-deaza-8-aza-dG, 2-amino-dA, and 2-thio-dT.

[0010] As a further improvement to a CpG oligonucleotide of the present invention, there is a homogeneous, heterogeneous or chimeric backbone in the sequence. Other bonds that can provide higher stability for nucleic acids of the present disclosure include but are not limited to dithiophosphate bonds, methylphosphonate bonds, methylthiophosphate bonds, boron phosphonate bonds, peptide bonds, alkyl bonds and dephosphorylation bonds. Therefore, in some embodiments, the CpG oligonucleotide has a non-natural backbone. Even, the CpG oligonucleotide has a backbone that is completely phosphorothioate.

[0011] As a further improvement of the CpG oligonucleotide described in the present invention, the sequence comprises modification of one or more than one phosphate groups.

[0012] As a further improvement of the CpG oligonucleotide described in the present invention, more than 50%, more than 60%, more than 70%, more than 80%, more than 90% of the nucleotides in the sequence are modified with phosphate groups.

[0013] As a further improvement of the CpG oligonucleotide described in the present invention, the modification of the phosphate group comprises one or more of a phosphorothioate internucleotide bond, a methylphosphonate bond and a boranephosphate bond.

[0014] Beneficial effects of this application:

[0015] The CpG oligonucleotides of the present invention include, but are not limited to, modifications of 3'OH or 5'OH groups, modifications of nucleotide bases, modifications of sugar components, modifications of backbones and modifications of phosphate groups. Such modifications can make CpG oligonucleotides more stable and / or less susceptible to degradation under certain conditions. For example, in some embodiments, CpG oligonucleotides are resistant to nucleases. Such modifications can be performed during the synthesis of oligonucleotides or after synthesis, and the modifications can occur on the phosphodiester bridges between nucleosides, on the ribose units and / or on natural nuclear bases (i.e., adenine, guanine, cytosine and thymine). When modified during the synthesis of oligonucleotides, the modified bases can be incorporated into the interior of the oligonucleotide or located at the end of the oligonucleotide. When modified after the synthesis of oligonucleotides, the modifications can be performed using active groups, for example, by amino-modified components, by 3' or 5'OH, or by phosphate groups. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The CpG TY001 and CpG TY002 provided by the present invention have mouse TLR9 activation effect;

[0017] Figure 2 is a schematic diagram showing that CpG TY001 and CpG TY002 effectively stimulate human PBMC to secrete IFN-α;

[0018] Figure 3 is a schematic diagram showing that CpG TY001 and CpGTY002 effectively stimulate human PBMC to secrete IL-6;

[0019] Figure 4 is a schematic diagram showing that CpG TY001 and CpGTY002 effectively stimulate human PBMC to secrete TNF-α;

[0020] Figure 5 It is a schematic diagram showing that CpG TY001 and CpGTY002 effectively stimulate human PBMC to secrete IFN-γ. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] serial number Nucleic acid sequence Length (bp) CpGTY001 5'-TCGCTTCGAAGCCAACGTTGG-3' 21 CpGTY002 5'-TCGCAACGTTGCCTCGAAGG-3' 20 CpG7909 (positive control) 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' 24 CpG1018 (positive control) 5'-TGACTGTGAACGTTCGAGATGA-3' 22 GC (negative control) 5'-TGGCCAAGCTTGGGCCCCTTGCAAGGGCC-3' 29

[0023] Table 1 Artificially synthesized single-stranded deoxyoligonucleotides containing CpG

[0024] Example 1. Preparation of artificially synthesized CpG-containing single-stranded deoxyoligonucleotide (solid phase synthesis method)

[0025] 1. Reagents and Materials

[0026] DBL (dichloroacetic acid toluene solution), 5-ethylthiotetrazolyl, diphenylacetyl disulfide, diethylamine, ammonia water, acetonitrile, acetic anhydride, 1-methylimidazole, pyridine, 3-methylpyridine, solid phase support, monomers A, G, C, T.

[0027] 2. Instruments and Equipment

[0028] Autooligo Pilot nucleic acid synthesizer, electronic balance, constant temperature water bath, diaphragm vacuum pump, medical refrigerator, medical low-temperature storage box.

[0029] 3. Methods

[0030] De-dimethoxytrityl reaction: 10% dichloroacetic acid toluene solution was used to remove the dimethoxytrityl (DMT) on the carrier and the 5' end of the nucleoside to obtain the 5' hydroxyl end to allow chain extension from the 3' end to the 5' end, providing a reaction site for the next reaction;

[0031] Coupling reaction: Under nitrogen protection, the equipment simultaneously introduces the phosphoramidite-protected nucleoside monomer and a certain proportion of activator into the synthesis column to react, first obtaining a 3'-terminal activated nucleoside phosphite-activated intermediate, which then undergoes a condensation reaction with the 5' hydroxyl group in the solid phase carrier;

[0032] Thio-reaction: inject 2 column volumes of thio-reagent, reaction time 1-6min, oxidize and thiolate the phosphite triester into a stable thiophosphate triester (i.e., trivalent phosphorus is sulfided into pentavalent phosphorus);

[0033] Capping reaction: There may be very few 5'-hydroxyl groups that do not participate in the condensation reaction. In this case, use capping reagent A and capping reagent B (1:1) for a total of 0.5 column volume and a reaction time of 0.6-2min to cap the 5'-hydroxyl groups that do not participate in the reaction to avoid continued reaction in the next one or more cycles and increase in N-impurities. The capped short fragments are easily separated during the purification stage.

[0034] Decyanoethylation reaction: Use 20% diethylamine solution, reaction time 5-20 min, flow rate 50-75 cm / h to remove the cyanoethyl group on the phosphate bond.

[0035] Aminolysis: The phosphite bond is connected to the oligonucleotide attached to the controllable solid phase carrier, and the phosphite bond is unstable and easily hydrolyzed by alkali. After the synthesis is completed, the solid phase carrier is drained and added to the hydrogenation reaction bottle. According to the feeding amount: add ammonia water, seal and shake evenly. Place it at 55°C in a constant temperature device for 1-24h. After the reaction solution cools to room temperature, filter the carrier and collect the filtrate in a reagent bottle, then rinse the carrier three times with purified water and collect the filtrate in the same bottle, and finally dilute to a fixed volume (rounded to the nearest 1.2 to 3.0 times the amount of ammonia water feeding) to obtain a stable CpG single-stranded deoxy oligonucleotide.

[0036] Example 2: HEKBlu TM e mTLR9 cell detection of biological activity of CpG ODN

[0037] By detecting HEK-Blue TM The secretion of embryonic alkaline phosphatase (SEAP) in the mTLR9 cell line was used to evaluate the activation effect of different concentrations of CpG on mouse TLR9.

[0038] HEK-Blue was stimulated with certain concentrations of CpG TY001, CpG TY002, CpG1018, CpG7909, and GC (non-CpG) TM mTLR9 cells (expressing mouse TLR9) activate NF-κB and AP-1, thereby inducing the production of SEAP, which can be expressed by Quanti-Blue TM Reagents to detect, Quanti-Blue TM The reagent binds to SEAP and displays a blue color. The OD value at 650 nm is measured by a spectrophotometer to quantify the level of SEAP, thereby evaluating the activation of mouse TLR9 by different CpGs.

[0039] 1. Reagents and Materials:

[0040] HEK-Blue TM mTLR9 cells (MCB, master cell bank);

[0041] DMEM (Gibco, 11995-065); FBS (Gibco, 10099-141C); Zeocin TM (ant-zn-05, InvivoGen, USA); Blasticidin TM (ant-b1-05, InvivoGen, USA); Normocin TM (ant-nr-1, InvivoGen, USA), Pen-Strep (Gibco, 15140-122); Quanti-Blue TM (Invivogen, rep-qbl); 96-well plate (Costar, 3599).

[0042] Growth medium: containing 4.5 g / L glucose, 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum, 100 μg / mL Normocin TM , Pen-Strep (100U / mL-100μg / mL) DMEM medium. After the revived cells are passaged twice, the growth medium needs to be supplemented with the selective antibiotic 100μg / mL Zeocin TM and 30 μg / mL Blasticidin TM .

[0043] QUANTI-Blue TM Detection solution: Add 1 mL of QB reagent and 1 mL of QB buffer to a 250 mL sterile culture bottle, add sterile water to 100 mL, vortex to mix, and incubate at room temperature for 10 min before use.

[0044] Test sample solution: Weigh an appropriate amount of the ultrafiltrate of the test sample (CpG TY001, CpG TY002, CpG1018, CpG7909, GC (non-CpG)), dilute it to 1 mg / ml with ultrapure water, and then dilute it to the required concentration with growth medium without supplementation of selective antibiotics.

[0045] 2. Methods:

[0046] In an incubator at 37°C and 5% CO2, HEK-Blue TM The mTLR9 cell line was cultured in vitro as an adherent monolayer in growth medium supplemented with selective antibiotics. The medium was changed twice a week and cells were usually cultured at a rate of at least 2-3 × 10 5 The density of cells / mL was not more than 15 generations.

[0047] Take HEK-Blue in logarithmic growth phase TMmTLR9 cells were washed and digested to prepare single cells, and the cells were resuspended in fresh growth medium supplemented with selective antibiotics to adjust the cell density to 2-3×10 5 Pieces / mL.

[0048] Add the prepared cell suspension to a U-shaped 96-well plate at 180 μL / well. Place the culture plate in an incubator at 37°C, 5% CO2, and saturated humidity for about 1-24 hours, then fill each well with liquid to 180 μL. Add different concentrations of the sample solution to the corresponding wells of the U-shaped 96-well plate at 20 μL / well, with final concentrations of 60 μg / ml and 120 μg / ml, respectively, and continue to culture for 24 hours.

[0049] After the culture was completed, the U-shaped 96-well plate was centrifuged at 1000 rpm for 5 min. 40 μL of the supernatant was aspirated from each well and placed in a flat-bottomed 96-well plate. 160 μL of QUANTI-Blue was quickly added. TM The test solution was shaken for a while and placed in a 37°C, 5% CO2 incubator for further incubation for 10-30 min. The 96-well test plate was taken out and the OD value at a wavelength of 650 nm was measured using a spectrophotometer.

[0050] 3. Results:

[0051] See Figure 1 . Figure 1 Both sequences showed significant responses, which were positively correlated with the dose. The reverse sequence GC had no activating effect on mTLR9.

[0052] Example 3: Cytokine activity after stimulation of human PBMC with different CpGs

[0053] After normal human PBMCs were stimulated in vitro with different concentrations of different CpGs for 1-24 h, the levels of cytokines such as IFN-α, TNF-α, and IL-6 in the cell supernatant were measured to evaluate the immunostimulatory activity.

[0054] 1. Reagents and Materials:

[0055] RPMI-1640 (Gibco, 11875-093); FBS (Gibco, 10099-141C); Pen-Strep (Gibco, 15140-122); Ficoll-Paque TMPLUS (GE, 17144003-1); Human TNF-αPrecoated ELISA kit (Dayou, 1117202); Human IFN-αPrecoated ELISA kit (Dayou, 1110012); Human IL-6Precoated ELISA kit (Dayou, 1110602); Human IFN-γPrecoated ELISA kit (Dayou, 1110002); fresh healthy human whole blood.

[0056] Complete culture medium: RPMI-1640 culture medium containing 10% fetal bovine serum and 100 U / mL-100 μg / mL Pen-Strep.

[0057] 2. Methods:

[0058] Human PBMC isolation. Transfer 30 mL of peripheral blood to a 100 mL sterile bottle, add 30 mL of saline at a ratio of 1:1, and mix well. Take 10 15 mL sterile centrifuge tubes and add 5 mL of Ficoll-Paque TM PLUS, and slowly add 6mL of diluted blood on it, being careful not to damage the interface. Transfer to a centrifuge, centrifuge at 960g for 30min at room temperature. Pipette the buffy coat layer into a 50mL centrifuge tube, add 20mL of saline, centrifuge at 319g for 10min at room temperature. Discard the supernatant, and resuspend the cells in 40mL of saline, centrifuge at 460g for 5min at room temperature. Resuspend the cells in 5mL of complete medium, and adjust the concentration of the suspension to 1-2×10 according to the number of cells. 6 / mL for future use.

[0059] Cell seeding: Add the cells with adjusted concentration into a 96-deep-well plate at 900 μL / well according to the well plate layout design, and culture them in a 37°C, 5% CO2 incubator for 1-6 hours.

[0060] CpG ODN stimulation: dilute CpG ODN with complete medium and add 100 μL per well to the above 96 deep-well plate. No duplicate wells are set for a single concentration. The working concentrations of CpG ODN are 0, 0.25, 0.5, 2, 8, 32, and 128 μg / ml. Incubate in a 37°C, 5% CO2 incubator for about 11-24 hours.

[0061] ELISA cytokine content detection: According to the detection method provided in the instructions of each kit, the content of IFN-α, IL-6, TNF-α, and IFN-γ in the cell supernatant was detected.

[0062] 3. Results:

[0063] See Figure 2-5 ,like Figure 2 As shown in the figure, CpG TY001 has a strong stimulating effect on the ability of stimulating human PBMC to secrete IFN-α, CpG TY002 is also significantly better than CpG1018, and GC has no stimulating effect; Figure 3 As shown in the figure, when the concentration of CpG ODN is higher than 0.25 μg / ml, CpG TY001 and CpG TY002 have a very significant ability to stimulate secretion compared with CpG 1018, while GC has no stimulating effect. Figure 4 As shown, CpGTY001 and CpG TY002 were significantly stronger than CpG1018 in stimulating human PBMC to secrete TNF-α, while GC had no stimulatory effect; Figure 5 As shown, when the concentration of CpG ODN was higher than 2 μg / ml, CpG TY001 and CpG TY002 had a very significant ability to stimulate secretion of IFN-γ from human PBMC, which was better than CpG1018, while GC had no stimulating effect.

[0064] Although the embodiments of the present application have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present application, and that the scope of the present application is defined by the claims and their equivalents.

Claims

1. A CpG oligonucleotide, characterized in that This includes a sequence encoding CpG TY001 or a sequence encoding CpG TY002.

2. A CpG oligonucleotide according to claim 1, characterized in that: The CpG oligonucleotide includes modifications of 3'OH or 5'OH groups, modifications of nucleotide bases, modifications of sugar components, modifications of backbones and modifications of phosphate groups.

3. A CpG oligonucleotide according to claim 1, characterized in that: CpG oligonucleotides contain naturally occurring or modified non-naturally occurring bases, modified sugars, phosphates and / or termini.

4. A CpG oligonucleotide according to claim 1, characterized in that: CpG oligonucleotides contain one or more ribonucleotides, deoxyribonucleotides, modified sugars or sugar analogs.

5. A CpG oligonucleotide according to claim 1, characterized in that: CpG oligonucleotides have homogeneous, heterogeneous or chimeric backbones.

6. A CpG oligonucleotide according to claim 1, characterized in that: The CpG oligonucleotides may contain one or more than one phosphate group modification.

7. A CpG oligonucleotide according to claim 1, characterized in that: More than 50%, more than 60%, more than 70%, more than 80%, more than 90% of the nucleotides in the CpG oligonucleotide are modified with phosphate groups.

8. A CpG oligonucleotide according to claim 6 or 7, characterized in that: The modification of the phosphate group comprises one or more of a phosphorothioate internucleotide bond, a methylphosphonate bond, and a boranophosphate bond.

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