Composition for treating intracellular infection and / or cancer

Abstract

The invention relates to the field of biological medicine, in particular to a composition for treating intracellular infection and / or cancer. The present invention provides a composition comprising (a) an ADA2 blocker; and (b) a TLR9 agonist. The composition provided by the invention can enhance the response of immune cells (such as pDC) to the TLR9 agonist, thereby realizing better treatment on intracellular infection and / or cancer.

Description

Priority Application

[0001] This application claims priority to PCT International Application PCT / CN2023 / 134255, "A composition for the treatment of intracellular infection and / or cancer", filed on November 27, 2023, the entire priority invention patent application of which is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention relates to the field of biomedicine, and specifically relates to a composition for the treatment of intracellular infection and / or cancer. Background Art

[0003] Adenosine is an important signaling molecule and the main nucleoside metabolite of the purine salvage pathway. Adenosine concentration is regulated by adenosine deaminase (ADA). Humans have two types of ADA: ADA1 and ADA2. ADA1 converts (deoxy)adenosine to (deoxy)inosine in the cytoplasm, which is crucial for cell survival. Genetic deficiency states that affect ADA1 expression lead to lymphocyte toxicity and a sharp decrease in B cells and T cells, manifested as severe combined immunodeficiency (SCID). Different from ADA1, the function of the second adenosine deaminase, ADA2, is still unclear. ADA2 is a secreted protein, and its ADA catalytic activity is much lower than that of ADA1 under the detection of physiological concentrations of adenosine. This latter observation, along with the relative lack of deoxyadenosine deamination, suggests that ADA2 may have another function different from ADA activity. Gene expression analysis shows that ADA2 is highly expressed in monocytes, myeloid cells, and plasmacytoid dendritic cells (pDC). NK cells, T cells, and B cells also express low levels of ADA2. The concentration of ADA2 in some biological fluids increases with the activation of immune cells. Several studies have shown that ADA2 is an important and reliable biomarker for diagnosing or monitoring treatment responses, such as pleural tuberculosis, HIV, autoimmune diseases, and various cancers, etc. Summary of the Invention

[0004] In a first aspect, the present invention provides a composition, which comprises:

[0005] (a) an ADA2 blocker; and

[0006] (b) a TLR9 agonist;

[0007] wherein the ADA2 blocker comprises one or more of a gene tool for reducing ADA2 expression, linear RNA, and IL-3;

[0008] the TLR9 agonist is CpG oligodeoxynucleotide (CpG ODN).

[0009] In some embodiments, the length of the TLR9 agonist is at least 20 nucleotides.

[0010] Toll-like receptor TLR9 is one of the intracellular TLR receptors that can respond to bacterial and viral infections, autoimmune diseases, and cancer. Human TLR9 is expressed and active in pDCs, B cells, and NK cells. The data of the present invention show that ADA2 can act as a molecular switch to control the activation of TLR9. Specifically, ADA2 can bind to class B and class C CpG ODNs and can thus compete with TLR9 for binding. In addition, the data of the present invention also show that ADA2 can also bind to RNA, particularly RNA molecules with a linear structure (whereas TLR9 does not bind to RNA). Using natural or chemically modified linear RNA can block the binding of the TLR9 agonist to ADA2 (the binding of ADA2 to the TLR9 agonist is stronger), to activate the pDC response to the TLR9 agonist and thereby increase the secretion of IFN-α.

[0011] In some embodiments, the nucleotides in the linear RNA are linked by phosphodiester bonds or phosphorothioate bonds.

[0012] In some embodiments, the length of the linear RNA is at least 15 nucleotides. In some embodiments, the length of the linear RNA includes 20 - 40 nucleotides.

[0013] In some embodiments, the linear RNA includes a poly U sequence.

[0014] Similarly, the data of the present invention show that reducing the expression of ADA2 by a gene tool that reduces ADA2 expression (such as siRNA) enhances the activation of TLR9.

[0015] In some embodiments, the gene tool that reduces ADA2 expression includes ADA2-specific siRNA.

[0016] In some embodiments, the ADA2-specific siRNA includes a sequence as shown in SEQ ID NO:20 or SEQ ID NO:21 or SEQ ID NO:22.

[0017] Similarly, the data of the present invention also show that IL-3 can also reduce the expression of ADA2, thereby enhancing the activation of TLR9.

[0018] In some embodiments, the TLR9 agonist is a class A CpG oligodeoxynucleotide. Class A CpG ODNs have a palindromic sequence containing CpG dinucleotides as the core, with poly G tails at both ends and a phosphorothioate-modified phosphodiester bond backbone.

[0019] In some embodiments, the class A CpG oligodeoxynucleotides include ODN 2216 or ODN 2336.

[0020] In some embodiments, the TLR9 agonist is a class B CpG oligodeoxynucleotide. Class B CpG ODNs are fully phosphorothioated linear CpG ODNs.

[0021] In some embodiments, the class B CpG oligodeoxynucleotides include one or more of ODN 2006PTO, ODN BW006 PTO, and ODN D-SL01 PTO.

[0022] In some embodiments, the TLR9 agonist is a class C CpG oligodeoxynucleotide. Class C CpG ODNs are fully phosphorothioated CpG ODNs that can form dimers through palindromic sequences and have the activities of both class A and class B CpG-ODNs.

[0023] In some embodiments, the class C CpG oligodeoxynucleotides include one or more of ODN D-SL03 PTO, ODN M362PTO, and ODN 2395PTO.

[0024] In a second aspect, the present invention provides the use of the above-mentioned composition in the preparation of a medicament for treating intracellular infections and / or cancers. The present invention demonstrates that ADA2 is a molecular switch that controls TLR9 activation in pDCs. After treatment with the above-mentioned ADA2 blocker, pDCs can enhance the response to the TLR9 agonist, thereby treating intracellular infections and / or cancers.

[0025] In some embodiments, the use includes enhancing the immune response against the intracellular infection and / or cancer.

[0026] In some embodiments, the ADA2 blocker is administered before treating with the TLR9 agonist.

[0027] In some embodiments, the ADA2 blocker is used to enhance the response of immune cells (such as plasmacytoid dendritic cells (pDCs)) to the TLR9 agonist.

[0028] In some embodiments, the use includes enhancing the secretion of IFN-α by plasmacytoid dendritic cells.

[0029] In some embodiments, the intracellular infection includes bacterial infection or viral infection.

[0030] In some embodiments, the cancer includes solid tumors and hematological tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0032] Figure 1 It is a graph of the experimental results of the binding of ADA2 to dsDNA;

[0033] Figure 2 It is a graph of the experimental results of the inhibition of the binding of ADA2 to ODN 2006PTO biotin;

[0034] Figure 3 It is a graph of the experimental results of the inhibition of the binding of ADA2 to ODN 2006PD biotin;

[0035] Figure 4 It is a graph of the experimental results of the binding of ADA2 to ODN 2006PTO in macrophage lysosomes;

[0036] Figure 5 It is a graph of the experimental results of the activation of pDCs by 3 types of ODNs PTO;

[0037] Figure 6 It is a confocal microscopy result graph of pDCs under different treatments;

[0038] Figure 7 It is a graph of the experimental results of the effect of knocking down or blocking ADA2 on TLR9 activation in pDCs;

[0039] Figure 8 It is a schematic diagram of the regulatory mechanism of ADA2 activating TLR9 in human pDCs;

[0040] Figure 9 It is a graph of the experimental results of the effect of IL-3 on human plasmacytoid dendritic cells (pDCs) after 48 hours of cell culture;

[0041] Figure 10 It is a schematic diagram of human pDCs releasing IFN-α and IL-8 after TLR9 activation by class A and class B ODN PTO;

[0042] Figure 11 It is a schematic diagram of the binding of ADA2 to ODN;

[0043] Figure 12 It is a graph of the experimental results of the binding of mouse Fc-TLR9 to ODN PTO;

[0044] Figure 13Results graph of the concentration of ADA2 in GM-CSF-differentiated monocytes intracellularly and in the cell culture medium 6 days after differentiation;

[0045] Figure 14 Experimental results graph of the localization of TLR9 in monocyte-derived macrophages;

[0046] Figure 15 Confocal microscopy results graph of 293T cells overexpressing ADA1 and ADA2. Detailed implementation manners

[0047] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0048] As used herein, "and / or" includes any and all combinations of one or more of the listed related items.

[0049] As used herein, "a plurality" means two or more, that is, it includes two, three, four, five, etc.

[0050] It should be noted that, as used herein, the term "comprise", "include" or any other variant thereof is intended to cover a non-exclusive inclusion, such that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the phrase "comprising a..." does not exclude the presence of additional identical elements in the process, method, article or device comprising that element.

[0051] As used in this specification, the term "about" typically represents + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0052] In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be understood that this type of "within a certain range" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Accordingly, the description of the range should be considered to have specifically disclosed all possible sub-ranges and the individual numerical values within that range. For example, the description of the range from 1 to 6 should be regarded as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0053] Detailed Description of the Drawings

[0054] Figure 1 :(A) Gel shift analysis of the binding of ADA2 (μg / ml) to plasmid DNA (μg / ml) with increasing salt concentration (mM). (B) Gel shift analysis of the binding of ADA2 (μg / ml) to plasmid DNA (μg / ml) in the presence of different concentrations of DNAse I (IU / ml). (C) Gel shift analysis of the binding of ADA2H88G and ADA2 (μg / ml) to plasmid DNA (μg / ml) in the presence and absence of different concentrations of ODN 2006PTO (μM). (D) Binding of ADA2 to Escherichia coli DNA, analyzed by DNA enzyme-linked immunosorbent assay. Each point represents the average of three replicates.

[0055] Figure 2 :(A) Inhibition of the binding of ADA2 to ODN 2006PTO biotin by class B ODN 2006PTO, ODN 2006GC PTO, class A ODN 2216PTO, and ODN 2336PTO. (B) Inhibition of the binding of ADA2 to ODN 2006PTO biotin by class B ODN BW006 PTO, ODN D-SL01 GC PTO, and class C ODN 2395PTO, ODN M362 PTO, and ODN D-SL03 PTO. (C) Binding of ADA2 to ODNs PTO (ODN 2006PTO and ODN 2216PTO) and ODNs PD (ODN 2006PD and ODN 2006-2006PD). (D) Binding of ADA2 to ODN 2006PD in buffers of different pH. Each point represents the average of three replicates.

[0056] Figure 3: (A) Inhibition of the binding of ADA2 to biotinylated ODN 2006PD by class B (ODN 2006PD), class A (ODN 2216PD and ODN 2336PD), and class C (ODN 2395PD) ODNs. (B) Inhibition of the binding of ADA2 to biotinylated ODN 2006PD by ODN 2006PD, ODN GC2006 PD, ODN 2006C-Met PD, poly T(24), ODN 2006Reverse PD, ODN 2006(T to C)PD. (C) Inhibition of the binding of ADA2 to biotinylated ODN 2006PD by modified class A ODN 2336PD. (D) Inhibition of the binding of ADA2 to biotinylated ODN 2006PD by poly T of different lengths. (E) Inhibition of the binding of ADA2 to biotinylated ODN2006PD by RNA PTO and RNA PD. (F) Figure 3 The oligonucleotide sequences used in Figure 3 (Table 2). Each point represents the mean of three replicates.

[0057] Figure 4 : (A, B) ADA2 levels in cell lysates (A) and culture supernatants (B) of macrophages cultured with 0.5 μM ODNs PTO for 24 h. Monocytes were isolated from PBMCs and differentiated into macrophages with 40 ng / ml GM-CSF for 6 days. Macrophages were rinsed twice with PBS before lysis to remove secreted ADA2. ADA2 levels were measured by enzyme-linked immunosorbent assay. Error bars represent the standard deviation of four independent measurements. (C-F) Confocal microscopy of macrophages differentiated with 40 ng / ml GM-CSF for 6 days and treated with 0.5 μM ODNs for 24 h. Cells were fixed and stained with ADA2 (blue), lysosome marker LAMP2 (red), nuclear marker DAPI (white), and endoplasmic reticulum marker ER-tracker (red). The ODNs used were ODN 2006PTO (D), ODN 2006G5 PTO (E), or ODN 2006PTO FITC (F). Untreated cells are shown in (C).

[0058] Figure 5 : (A) On day 1, 1 μM ODNs PTO was added to pDCs (0.0125x10 6 cells / ml), and the amounts of IFN-α (A) and IL-8 (B) released by the cells were analyzed by ELISA on the next day. Class A ODNs are shown in blue, class B in red, and class C in green. (C, D) On day 1 or day 2, class A ODN 2216PTO and class B ODN 2006PTO (1 μM) were added to pDCs (0.0125x10 6cells / ml), and the amount of IFN-α (C) or IL-8 (D) released by the cells was analyzed by ELISA on the next day. (E, F) ODNs PTO (1 μM) was added to pDCs (0.05x10 6 cells / ml) on day 1, and the amount of IFN-α released by the cells was analyzed by ELISA on the next day. Class A ODNs are shown in blue, Class B in red, and Class C in green. Error bars represent standard deviation, and the results are from four independent replicates.

[0059] Figure 6 : (A) Human pDCs were isolated from PBMCs and cultured in 8-well chambers with 10 ng / ml IL-3. After 24 hours, the pDCs attached to the plastic were washed, fixed, and stained with anti-ADA2 antibody (blue), ERtracker (red) for endoplasmic reticulum staining, and DAPI (white) for nuclear staining. (B) Human pDCs were cultured in 8-well chambers with 10 ng / ml IL-3 in the presence of 0.5 μM ODN 2006PTO FITC. After 24 hours, the pDCs attached to the plastic were washed, fixed, and stained with anti-LAMP2 antibody (red), anti-ADA2 antibody (blue), and DAPI (white). (C) ODN 2006PTO FITC co-localized with ADA2 and the lysosomal marker LAMP2.

[0060] Figure 7 : In all experiments, human pDCs were isolated from PBMCs and cultured in the presence of 10 ng / ml IL-3. (A-D) On day 1, pDCs (0.0125x10 6 cells / ml) were transfected with siRNA using Lipofectamine RNAiMax transfection reagent. On day 2, ODNs PTO (1 μM) was added to the cells. On day 3, the amounts of IFN-α (A, C) and IL8 (B, D) in the cell culture medium were analyzed using ELISA. (E, F) Knockdown of ADA2 expression in pDCs with siRNA increased the activation of TLR9 by dsDNA. Cells (0.0125x10 6cells / ml). After 24 h, (E) Escherichia coli genomic DNA and (F) human THP1 cell genomic DNA were transfected with Lipofectamine 3000. (G) ADA2 in the culture supernatant of siRNA-transfected PMA-activated THP1 cells was analyzed by ELISA 2 days later. (H) Poly U was used to block the activation of TLR9 by ADA2-increasing ODN 2006PTO. On day 1, human pDCs were transfected with 0.2 μg Poly U or Poly U21 using LipofectamineRNAiMax transfection reagent (0.02x10 6 cells / ml). On day 2, ODN 2006PTO (0.5 μM) was added to the cells. On day 3, the amount of IFN-α in the cell culture supernatant was analyzed by ELISA. Error bars represent standard deviation, and the results are from four independent replicates. In all experiments, the differences between the siRNA control and siRNA ADA2 were statistically significant (p < 0.008).

[0061] Figure 8 : (A) CpG ODN 2006 was added on the first day, and pDCs were freshly isolated. (B) CpG ODN 2006 was added on the second day, and pDCs were those incubated with IL-3 for 24 h. Endosome, endosome; Lysosome, lysosome.

[0062] Figure 9 : Within the first 24 h, pDCs were cultured with 10 ng / ml IL-3 in 8-well chambers. Thereafter, the cells were either left untreated (A - B) or re-treated with 0.5 μM ODN 2006PTO FITC for 24 h (C). Then, the pDCs attached to the plastic were washed, fixed, and stained with anti-ADA2 antibody (blue), ER-tracker (red), and DAPI (white), anti-LAMP2 antibody (green and red), and anti-CD303 as a pDC cell surface marker.

[0063] Figure 11 : In A, ODN 2006PTO was conjugated with streptavidin to capture ADA2, and then adenosine was added to detect ADA2 bound to the ODN. In B, ADA2 was captured using anti-ADA2 rabbit polyclonal antibody, and ODN2006PTO biotin was detected using HRP-streptavidin.

[0064] Figure 12: (A) Class A (ODN 1585PTO) and Class B (ODN 2006PTO, ODN 1826PTO, ODN 1668) inhibited the binding of murine Fc-TLR9 to ODN 2006PTO. (B) The binding of murine Fc-TLR9 to ODN2006PTO in the presence of increasing concentrations of ADA2. (C) The binding of murine Fc-TLR9 and ADA2 to ODN 2006PTO. Each point represents the mean of three replicates.

[0065] Figure 13 : Monocytes were isolated from PBMCs (0.4x 10 6 cells / ml) and differentiated into macrophages for 6 days with 40 ng / ml GM-CSF. Cells were washed with fresh medium and left untreated or treated with 0.5 μM CpG ODN 2006 or CpGODN 2006G5 for 24 hours. The concentration of ADA2 in cell lysates (A, intracellular ADA2) or cell culture medium (B, secreted ADA2) was analyzed by ELISA.

[0066] Figure 14 : Monocytes were obtained from PBMCs and differentiated into macrophages for 6 days using 40 ng / ml GM-CSF. The macrophages were then exposed to 0.5 μM ODN 2006PTO for 24 hours. After fixation, the cells were stained with anti-LAMP2 (green) and anti-TLR9 (blue) antibodies. Both (A) and (B) showed no co-localization of TLR9 with the lysosomal marker LAMP2.

[0067] Figure 15 : (A) 293T cells expressing ADA2 and GFP were fixed and stained with anti-LAMP2 (red) and anti-ADA2 antibody (blue). (B) 293T cells expressing ADA1 and GFP were fixed and stained with anti-LAMP2 (red) and anti-ADA2 antibody (blue). (C) 293T cells expressing ADA1 and GFP were fixed and stained with anti-LAMP2 (red) and anti-ADA1 antibody (blue).

[0068] Example 1: Materials and Methods

[0069] 1.1 Cell Isolation and Culture

[0070] Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood of healthy donors according to an IRB-approved protocol. Isolation was performed using 50 mL LeucosepTM tubes (Greiner Bio-One). After washing twice with 50 ml PBS buffer, CD14+ monocytes were purified using anti-CD14 conjugated magnetic beads (Miltenyi). pDCs were purified by negative or positive selection using the corresponding kits from Miltenyi. The cell purity was greater than 90% as determined by flow cytometry. In cell culture, RPMI 1640 was used as complete medium, supplemented with 1% non-essential amino acids, 1% sodium pyruvate, 100 U / ml penicillin, 100 μg / ml streptomycin, 2 mM L-glutamine and 10% FBS. Monocytes and pDCs were cultured in 96-well plates with 200 μl RPMI medium containing 40 ng / ml GM-CSF (PeproTech) or 10 ng / ml IL-3 (PeproTech) respectively. The complete medium containing 40 ng / ml GM-CSF was changed every three days. Monocytes were differentiated for 6 days and then incubated overnight with 0.5 μM CpG ODNs PTO (Invivogen) to analyze the intracellular binding of ADA2 to CpG ODNs PTO. Additionally, for microscopic analysis, cells were treated with 0.5 μM FITC-labeled CpG ODN 2006PTO (Invivogen).

[0071] 1.2 Knockdown of ADA2 expression in THP-1 cells and pDCs with siRNA

[0072] THP-1 cells (0.3 x 10 6(cells / ml), and cultured in 200 μl of RPMI medium in a 96-well plate. The medium was changed daily, and the cells were transfected with siRNA (1 pmol / well) using Lipofectamine RNAiMax reagent according to the manufacturer's protocol (Invitrogen). The next day, the cell medium was changed, and the concentration of ADA2 in the medium was analyzed by ELISA. pDCs were transfected with 1 pmol siRNAs, 0.2 μg poly U (ssPolyU Naked, Invivogen), or poly U21 (Synbio Technologies) and cultured in RPMI medium containing 10 ng / ml IL-3 for 1 day. The next day, the cells were activated with 0.5 μM CpG ODNs PTO (Invivogen), or transfected with 0.1 μg of THP1-1 or Escherichia coli DNA using Lipofectamine 3000 transfection reagent (Invitrogen). The next day, the amounts of IL-8 and IFN-α secreted by the cells were analyzed by ELISA (Biolegend).

[0073] Among them, the sequence of the siRNA control is: rCrGrUrUrArArUrCrGrCrGrUrArUrA rArUrArCrGrCrGrU AT (SEQ ID NO:19); the sequence of siRNA A is: rGrGrArUrArArGrUrUrCrArUrAr GrCrArGrArUrGrUrGrG CT (SEQ ID NO:20); the sequence of siRNA B is: rGrGrCrArUrArCrArGrCrArU rCrCrGrArUrUrUrArArUrC TG (SEQ ID NO:21); the sequence of siRNA C is: rCrCrUrCrUrArArUrCrAr CrArGrCrUrUrArUrArArUrCrG GA (SEQ ID NO:22). Among them, the underlined part represents ribonucleotides, and the non-underlined part represents deoxyribonucleotides.

[0074] 1.3 Immunostaining and confocal microscopy

[0075] Monocytes and pDCs were cultured in 400 μl of RPMI medium in 8-well chamber slides (Thermofisher). Cells were fixed in 10% formaldehyde or 4% PFA for 10 minutes, washed three times with PBS, permeabilized in PBS containing 0.3% Triton X-100 (PBS-T) for 5 minutes, and then washed three times with PBS. The primary antibody diluted in PBS containing 1% BSA was incubated with the cells overnight at 4 °C, then washed three times with PBS, and incubated with the secondary antibody in PBS containing 1% BSA for 0.5 hours at room temperature. After washing three more times, the cells were mounted in 80% glycerol. The dilution of the LAMP2 antibody (Sino Biological, 13555-MM05) and the ADA1 antibody (Abcam, ab34677) was 1:100. The dilution of the ADA2, TLR9 (Abcam, ab259651), and CD303-FITC (Biolegend, 354208) antibodies was 1:50. Fluorescent staining was captured by a Leica SP8 confocal microscope. The polyclonal anti-ADA2 antibody was purified from the serum of rabbits immunized with recombinant ADA2 (antisera were produced by Sino Biological). To verify the specificity of the antibody, the polyclonal rabbit antibody was used as a negative control, and no staining was found. In addition, the nuclei and endoplasmic reticulum (ER) were stained with DAPI (Cell Signaling Technology) and ERtracker (Invitrogen) according to the manufacturer's instructions.

[0076] 1.4 Analysis of the binding of ADA2 to DNA and CpG ODN

[0077] Recombinant ADA2 and its mutant ADA2 H88G were overexpressed and purified in HEK 293T cells. For the gel shift experiment, as Figure 1As shown, ADA2 was incubated with ΔR 8.2 packaging plasmid DNA for 5 minutes at room temperature in the presence of different concentrations of salt, DNase I (NovoProtein), or CpG ODN 2006PTO (Invivogen). Then, 15 μl of the mixture was analyzed using 1% agarose gel (GenStar) containing GelRed (Biosharp). In another experiment, 96-well ELISA plates were coated with 1 mg / ml Escherichia coli DNA (Invivogen) in 100 μl of TBS containing 0.02% NaN3 overnight at 37°C. Then, the plates were washed three times with 200 μl of 1x PBS containing 0.05% Tween 20 and blocked with 200 μl of PBS containing 2% BSA and 0.02% NaN3 for 1 hour at room temperature. Next, 100 μl of the ADA2 dilution in 50 mM Tris (pH 6.8), 150 mM NaCl, 10 μM ZnCl2, and 0.02% NaN3 (buffer A) was added to the plates containing DNA, and the plates were then incubated with shaking for 1 hour at room temperature. After washing three times with buffer A, 2 mM adenosine was added in the same buffer, and after incubation at 37°C for 20 hours, the amount of ADA2 bound to DNA was determined by comparing the ADA activity of the ADA2 retained on the DNA with that of an ADA2 standard. A similar analysis was performed for the binding of ADA2 to CpG ODNs (Invivogen), but with a different DNA capture strategy ( Figure 11 A). ELISA plates were coated with 200 μl of PBS containing 2 mg / ml streptavidin (New England Biolabs) and 0.02% NaN3 overnight at 4°C. The plates were washed three times with 200 ml of PBS containing 0.05% Tween 20 and blocked with 200 ml of PBS containing 2% BSA. Then, 100 μl of PBS containing 20 nM biotinylated DNA was added to the wells and incubated with shaking for 30 minutes at room temperature. The binding of ADA2 was analyzed using the streptavidin-bound CpG ODN biotin plates as described above. To study the inhibition of the binding of ADA2 to CpG ODN biotin, 50 μl of the same buffer containing 80 nM ADA2 was added before adding 50 μl of the DNA / RNA dilution (SynbioTechnologies) in buffer A to the wells. The amount of ADA2 remaining in the wells was determined as described above. To study the pH-dependence of the binding of ADA2 to CpG ODN 2006PD, 10 mM sodium citrate buffer at different pH values and 150 mM NaCl were used as the binding and washing buffers.

[0078] In the experiment using CpG ODN PTO, a modified method of standard ELISA was used to study the binding of ADA2 to ODN. Figure 11 B). An ELISA plate (Greiner Bio-One) was coated with 100 μl of PBS containing 5 μg / ml rabbit anti-ADA2 polyclonal antibody and 0.02% NaN3 overnight at 4 °C. After washing 3 times with 200 μl of PBS-Tween 20 buffer and blocking with 200 μl of PBS containing 2% BSA for 1 hour, 100 μl of PBS containing 100 ng / ml ADA2, 10% FBS and 0.02% NaN3 was added to the wells. Incubate on a shaker at room temperature for 1 hour. Subsequently, wash 3 times with 200 μl of PBS-Tween 20, and add 100 μl of a mixture of 4 nM CpG ODN 2006PTO biotin in buffer A and different concentrations of inhibitory CpG ODNs PTO to the wells. After adding 100 μl of 1:1000 streptavidin-HRP (Abbkine) in 20 mM Tris HCl (pH 6.8), 50 mM NaCl, 10 μM ZnCl2 (buffer B) containing 10% FBS, incubate on a shaker at room temperature for 30 minutes, and then wash 4 times with the same buffer. Add 100 μl of TMB substrate (BioLegend) to start the color reaction. Add 100 μl of 2 M HCl to stop the reaction and read at 450 nM on an ELISA reader (Thermofisher).

[0079] 1.5 mT TLR9 binding to CpG ODN 2006PTO

[0080] An ELISA plate (Greiner Bio-One) was coated with 100 μl of PBS containing 5 μg / ml goat anti-mouse antibody (Sinobiological) and 0.02% NaN3 overnight at 4 °C. After washing the plate 3 times with 200 μl of PBS-Tween 20 buffer and blocking with 200 μl of PBS containing 2% BSA for 1 hour, 100 μl of PBS containing 100 ng / ml Fc-mTLR9 (R&D Systems), 10% FBS and 0.02% NaN3 was added to the wells. Incubate on a shaker at room temperature for 1 hour. Subsequently, wash the plate 3 times with 200 μl of PBS-Tween 20, and add 100 μl of a mixture of 4 nM CpG ODN 2006 biotin in buffer A and different concentrations of inhibitory CpG ODN to the wells. In Figure 10In the experiment, after adding 50 μl of ADA2 at the indicated concentration to the Fc-mTLR9 plate, 50 μl of CpG ODN 2006 biotin with increasing concentration was added. 100 μl containing 1:1000 streptavidin-HRP (Abbkine), 20 mM Tris HCl (pH 6.8), 50 mM NaCl, 10 μM ZnCl2, 10% FBS (buffer B) was added to the wells, and then washed 4 times with the same buffer and incubated on a shaker at room temperature for 30 minutes. 100 ml of TMB substrate (BioLegend) was added to start the color reaction. 100 ml of 2 M HCl was added to stop the reaction, and then the absorbance at 450 nM was read on an ELISA reader (Thermofisher).

[0081] Example 2: Binding of ADA2 to dsDNA

[0082] First, the interaction between ADA2 and ΔR 8.2 packaging plasmid DNA was studied using the gel shift assay. As Figure 1 shown in A, the plasmid migrated in agarose gel in linear and supercoiled forms (lane 1). However, the addition of ADA2 resulted in the formation of a complex between the enzyme and the plasmid (lane 2). In addition, the ADA2 and plasmid complex was stable at physiological salt concentrations ( Figure 1 A). In addition, ADA2 bound to the plasmid protected the plasmid DNA from cleavage by DNase I ( Figure 1 B). The binding of ADA2 to the plasmid was independent of ADA activity, since the ADA2 H88G mutant with 100-fold lower ADA activity could also form a complex with the plasmid ( Figure 1 C). The addition of CpG ODN 2006 PTO could competitively disrupt the complex with plasmid DNA, indicating that CpG ODN 2006 PTO binds to ADA2 ( Figure 1 C). The experiment also demonstrated that ADA2 formed a complex with Escherichia coli DNA adsorbed on the ELISA plate ( Figure 1 D). These results indicate that under physiological conditions, ADA2 can interact with dsDNA and ODNs PTO independently of its ADA activity.

[0083] Example 3: Binding of ADA2 to ssDNA

[0084] Toll-like receptor TLR9 is an intracellular dsDNA sensor activated by unmethylated ODNs containing CG motifs. Phosphodiester oligodeoxynucleotides (PD ODNs) are usually modified with phosphorothioate (PTO) backbone to protect ODNs from degradation by DNases. Next, the binding of ADA2 to three classes of CpG ODNs PTO, which are used to activate TLR9 receptor in different experimental systems, was investigated.

[0085] The enzyme was captured on an ELISA plate with an anti-ADA2 antibody ( Figure 11 B). Then biotinylated CpG ODN 2006PTO of class B was added to ADA2 with increasing concentrations of different classes of CpG ODNs PTO ( Figure 2 ). In a standard ELISA, streptavidin-conjugated horseradish peroxidase (HRP) was added and then color development reaction was carried out with HRP substrate TMB, and the amount of biotinylated CpG ODN 2006PTO bound to ADA2 could be observed. Table 1 lists the ODN sequences and the obtained inhibition constants (IC50) in this experiment. The binding and washing buffers contained a near-physiological salt concentration of 150 mM and a pH value of 6.8, similar to primary endosomes. CpG ODN 2006PTO and its control GpC ODN 2006PTO (ODN 2006GC PTO) had the highest affinity for ADA2. They effectively inhibited the binding of biotinylated CpG ODN 2006PTO to the enzyme ( Figure 2 A, Table 1). In contrast, class A CpG ODNPTO (2216 and 2336) did not bind well to ADA2 ( Figure 2 A). Other CpG ODN PTO of class B and C bound to ADA2, but had a lower affinity for ADA2 than CpG ODN 2006PTO ( Figure 2 B). Except for CpG ODN 2006PTO, most CpGODN PTO could form secondary structures in solution, which might reduce their affinity for ADA2. In addition, the backbone of class A CpGODNs PTO was not completely modified with phosphorothioate, which also reduced their electrostatic binding force with ADA2.

[0086] Table 1 (Bases in capital letters are phosphodiesters, bases in lowercase letters are phosphorothioates (anti-nucleases), and underlines indicate palindromic sequences)

[0087] In a similar experiment, the affinity of murine TLR9 for CpG ODN 2006PTO was higher than that for other CpG ODNsPTO, while the affinity for class A CpG ODN PTO was very low( Figure 12 A). ADA2 added to murine TLR9 competitively bound to CpGODN 2006PTO biotin and completely eliminated the binding of murine TLR9 to CpG ODN 2006PTO biotin at high ADA2 concentrations( Figure 12 B). The relative affinity of ADA2 for CpG ODN 2006PTO was also higher than that of TLR9( Figure 12 C).

[0088] Next, the binding of ADA2 to native PD ODNs with and without PTO modification was compared. In this experiment, biotinylated ODN was first bound to streptavidin adsorbed on an ELISA plate( Figure 11 A). In the second step, ADA2 was added to the wells on the plate, and the ADA2 bound to the ODN was detected using its ADA activity. As Figure 2 shown in C, ADA2 bound to CpG ODN 2006PD, but the stability of this complex was lower than that of CpG ODN 2006PTO, with apparent Kd values of 13 and 4 nM, respectively. Doubling the length of CpG ODN 2006PD (ODN 2006-2006PD) did not change the apparent binding constant (Kd = 14 nM), but the number of binding sites tripled. These results indicate that more than one molecule of ADA2 binds to ODN 2006-2006PD. In addition, the binding of ADA2 to CpG ODN 2006-2006PD increased under low pH conditions, which is similar to the reported pH values of endosomes and lysosomes( Figure 2 D).

[0089] To further explore the interaction of ADA2 with native ODNs PD, the binding of ADA2 to CpG ODN 2006PD was monitored in the presence of increasing concentrations of competing ODNs( Figure 3 ). Similar to the modified ODNs PTO, ADA2 had the highest affinity for class B CpG ODN 2006PD compared to class C CpG ODN2395 and class A CpG ODNs 2216 and 2336. The binding of ADA2 to class A CpG ODN 2336PD was the weakest( Figure 3 A), indicating that the structure of the ODN is also crucial for the binding of ADA2 to native ODNs.

[0090] The ODN consisting of poly T has no secondary structure and can bind to ADA2 as well as CpG ODN 2006PDFigure 3 B). However, the change of thymidine to cytosine in CpG ODN 2006PD results in the formation of a secondary structure and a decrease in affinity. The methylation of cytosine and the change of CG to GC in the CpG ODN2006PD sequence reduce the binding constant of the modified ODN to ADA2 by 3-fold. Interestingly, after adding the complementary ODN 2006Reverse PD (forming a double strand with CpG ODN 2006PD), the binding of ADA2 to biotinylated CpGODN 2006PD completely disappears. This result indicates that ADA2 can bind better to ssDNA than to dsDNA.

[0091] To further demonstrate that the secondary structure affects the binding of ODNs PD to ADA2, the sequence of CpG ODN 2336PD, which binds poorly to ADA2, was modified ( Figure 3 C). After exchanging the nucleotides forming the palindrome with thymidine, the modified ODN binds better to ADA2. Meanwhile, the additional change between guanosine and thymidine at the 3'-end of the ODN does not affect the binding of the ODN to ADA2. Interestingly, changing four guanosines at the 5'-end of the ODN to thymidine reduces the binding of ADA2 to the ODN, indicating that the extension of guanosine at the 5'-end of the ODN may improve the binding of ADA2 to the ODN. The binding of ODNs PD to ADA2 depends on the length of the ODN. Studies on the binding of poly T to ADA2 show that the affinity of ADA2 for ODNs PD with a poly T length exceeding 12 increases until the ODN length reaches 24 nucleotides. ODNs with a length exceeding 24 nucleotides bind to multiple ADA2 molecules ( Figure 3 D).

[0092] It has been reported that TLR9 cannot recognize RNA. The present invention discovers that, different from TLR9, ADA2 can bind to RNA ( Figure 3 E). Compared with natural RNA (RNA PD), the enzyme has a higher affinity for RNA with a phosphorothioate backbone (PTO modification). In addition, similar to ODNs PD, the interaction of ADA2 with RNAs PD depends on their secondary structure, and linear Poly U is the best ligand compared with RNA 2006PD and RNA 40PD. Therefore, ADA2 can bind to ssDNA and RNA, and these interactions depend on the secondary structure of nucleic acids.

[0093] Other sequences used in this example are shown in Table 2.

[0094] Table 2

[0095] Example 4: ADA2 binds to ODN PTO in the lysosomes of macrophages.

[0096] Based on the above results, ADA2 binds to ODNs PTO within a range of salt concentrations and pH values, where the pH values are those reported for endosomes and lysosomes. Therefore, the following experiments were set up to confirm that ADA2 can bind to CpG ODNs PTO within cells. Monocyte-derived macrophages were cultured with CpG ODNs PTO, and the concentrations of ADA2 in the cell lysates and cell culture media were analyzed. The results showed that culturing macrophages with class B and class C CpG ODNs PTO led to an increase in intracellular ADA2 concentration. In contrast, intracellular ADA2 remained unchanged in the presence of class A CpG ODNs PTO( Figure 4 A). In addition, the amount of ADA2 secreted by the cells was not affected by CpG ODNs PTO( Figure 4 B, Figure 13 B), indicating that ADA2 interacts with ODNs within the lumen of endosomes. Therefore, macrophages were analyzed by confocal microscopy to determine the localization of ADA2.

[0097] Confocal microscopy of macrophages showed abundant intracellular expression of ADA2( Figure 4 C), and the enzyme was found to be partially co-localized with the lysosomal marker LAMP2. However, adding CpG ODN 2006PTO to macrophages increased the density of ADA2 within lysosomes( Figure 4 D). In contrast, treating cells with CpG ODN 2006PTO modified with a 3'-poly G string (ODN 2006G5 PTO) did not result in an increase in the density of ADA2 in lysosomes( Figure 4 E) or an increase in the intracellular enzyme concentration( Figure 13 A). In addition, ADA2 co-localized with fluorescently labeled CpG ODN 2006PTO FITC within this organelle( Figure 4 F). These observations explain the Figure 4 ELISA results in A and suggest that ADA2 bound to CpG ODN 2006PTO is transported to lysosomes. In addition, CpG ODN 2006PTO may protect ADA2 from digestion by lysosomal proteases, which may explain why the concentration of the intracellular enzyme increases. In contrast to ADA2, the density of the TLR9 receptor in lysosomes did not change after culturing macrophages with CpG ODN 2006PTO( Figure 14 ).

[0098] HEK293T cells overexpressing ADA2 also showed partial co-localization of ADA2 with the lysosomal marker LAMP2( Figure 15A). In contrast, ADA1 was not found in the lysosomes of cells overexpressing ADA1( Figure 15 C), indicating that ADA2 is the only ADA in lysosomes.

[0099] Example 5: After activation with IL-3, pDCs respond differently to three types of CpG ODNs PTO.

[0100] In humans, when unmethylated ODNs PTO containing CG motifs activate TLR9, pDC cells produce a large amount of IFN-α. Class A CpG ODNs PTO has the lowest affinity for TLR9( Figure 12 A), but can still induce freshly isolated cells to secrete a large amount of IFN-α. In contrast, the same cells produce more IL-8 in response to Class B CpG ODNs PTO. It has been reported that TLR9 activation in early endosomes leads to the secretion of IFN-α through the IRF-7 pathway. In contrast, the binding of TLR9 to CpG ODNs PTO in lysosomes induces the NF-κB pathway and the secretion of IL-8( Figure 10 ). The activation of TLR9 in early endosomes and lysosomes seems to be regulated differently.

[0101] The experimental results of this example show that the addition of Class A and Class C CpG ODNs PTO to freshly isolated pDCs leads to the secretion of IFN-α. The addition of Class B CpG ODNs PTO does not induce the release of a large amount of IFN-α( Figure 5 A). In the same experiment, compared with Class A ODNs PTO, Class B and Class C CpG ODNs PTO induced more IL-8 production in cells after activating TLR9( Figure 5 B). However, after culturing in a medium containing IL-3 for one day, when CpG ODNs PTO was added to pDCs, the response to different classes of CpG ODNs PTO changed. Here, the cells began to secrete IFN-α in response to Class B activation( Figure 5 C), while the secretion of IL-8 was significantly reduced( Figure 5 D). In addition, when the number of pDCs increased, the amount of IFN-α released by cells activated with Class B and Class C CpG ODNs PTO on the second day was much higher than that on the first day of activation( Figure 5 E, Figure 5 F). This pattern was similar to the secretion of IL-8 during TLR9 activation on Day 1( Figure 5 B). These results indicate that the three classes of CpG ODNs PTO have an intracellular regulatory effect on the secretion of IFN-α and IL-8 by pDCs during TLR9 activation.

[0102] Example 6: ADA2 can regulate the activity of TLR9 in pDCs.

[0103] Next, plasmacytoid dendritic cells (pDCs) isolated from peripheral blood mononuclear cells (PBMCs) were analyzed using a confocal microscope. It was found that these cells expressed ADA2 (as shown in Figure 6 A). However, there was no obvious co-localization of this enzyme with the lysosomal marker LAMP2. After culturing pDCs with IL-3 and CpG ODN 2006PTO FITC for one day, a large portion of ADA2 in the lysosomes co-localized with CpG ODN 2006FITC (as shown in Figure 6 B and Figure 6 C). This finding was similar to that previously observed in monocyte-derived macrophages, indicating that ADA2 bound to CpG ODN 2006 in the endosomes of pDCs was also transported to the lysosomes.

[0104] Notably, when pDCs were treated with CpG ODN 2006PTO on the second day of culturing pDCs with IL-3, no co-localization of ADA2 with CpG ODN was found (see Figure 9 ). The reason may be that the concentration of ADA2 in the endosomes decreased after treating pDCs with IL-3.

[0105] As shown in Figure 4 , ADA2 could bind to intracellular CpG ODNs PTO, indicating that it might compete with TLR9 for binding to class B CpG ODN 2006PTO in the endosomes. To further study this issue, the expression of ADA2 in pDCs was knocked down using siRNA, and then TLR9 was activated with CpG ODNs PTO or dsDNA (see Figure 7 ).

[0106] In this example, the efficiency of knocking out ADA2 using three different siRNAs and a scrambled siRNA control was verified in THP1 cells, monocytes expressing ADA2. It was found in this example that the amount of ADA2 secreted from PMA-activated THP1 cells transfected with siRNA A and siRNA C decreased by 70 - 80%, while the siRNA control and siRNA B did not significantly affect the release of ADA2 from the cells ( Figure 7 G).

[0107] Next, freshly isolated pDCs were transfected with the same siRNAs and activated with CpG ODN2006PTO one day after transfection. It was found in this example that knocking down the expression of ADA2 with siRNA A and siRNA C led to an 8-fold increase in the secretion of IFN-α from pDCs ( Figure 7A). Similarly, knockdown of the expression of ADA2 in pDCs activated with three types of CpG ODNs PTO increased the secretion of IL-8 ( Figure 7 B). In addition, knockdown of the expression of ADA2 increased the secretion of IFN-α and IL-8 in pDCs activated with three types of CpG ODNs PTO ( Figure 7 C and Figure 7 D). However, in the corresponding experiments, cytokine production was not observed with the control ODN GpC 2006 (data not shown). These results suggest that ADA2 regulates the activity of TLR9 by competing for binding to CpG ODNs PTO.

[0108] Next, pDCs were treated with siRNA after transfection with DNA isolated from Escherichia coli and human THP1 cells. After the expression of ADA2 was knocked down, IFN-α production by pDCs transfected with bacterial and eukaryotic dsDNA increased significantly ( Figure 7 E and Figure 7 F), indicating that ADA2 regulates the activation of TLR9 with modified CpG ODNs PTO and native dsDNA.

[0109] Unlike TLR9, ADA2 can bind RNA ( Figure 3 E). Thus, RNA that binds to ADA2 can block the enzyme, allowing ODNs PTO to activate TLR9. To test this hypothesis, pDCs were transfected with long-length poly U and 21-nucleotide poly U (Poly U21) before activation with CpG ODN 2006PTO. As Figure 7 shown in H, cells transfected with RNA did not secrete IFN-α. However, after adding CpG ODN 2006PTO to cells transfected with poly U or poly U21, the amount of IFN-α released by the cells increased 3-fold compared to cells not treated with RNA. These results suggest that knockdown of the expression of ADA2 with siRNA and blocking the binding of ADA2 to CpG ODNs PTO with RNA can activate TLR9, leading to increased secretion of IFN-α by pDCs.

[0110] Summary

[0111] The present invention discovers that ADA2 is a DNA-binding protein, and the data of the present invention indicate that there is competition between ADA2 and TLR9 for binding to ssDNA in pDC endosomes. These pathways are shown to regulate the secretion of IFN-α and pro-inflammatory cytokines to varying degrees in response to viral infections and cancer.

[0112] Functional analysis showed that pDCs treated with class A and C but not B CpG ODNs PTO secreted large amounts of IFN-α. In contrast, cells released more IL8 after activation of TLR9 by class B and C CpG ODNs( Figure 5 A, Figure 5 B). These results suggest that activation of TLR9 in early endosomes leads to secretion of IFN-α by pDCs, while activation of TLR9 in lysosomes activates the NF-kB pathway and releases IL-8 from cells( Figure 10 ). Interestingly, pDCs pretreated with IL-3 responded very differently to the three classes of CpG ODNs. After culturing with CpG ODNs PTO for 1 day, cells pretreated with IL-3 produced IFN-α in response to treatment with class B CpG ODN 2006PTO( Figure 5 C, Figure 5 E, Figure 5 F). pDCs incubated with IL-3 for 24 hours and then treated with CpG 2006ODN FITC did not show colocalization of ADA2 with ODN 2006, indicating a reduced concentration of ADA2 in activated pDCs( Figure 8 B). This further explains why pDCs cultured with IL-3 for one day started to produce IFN-α and released a smaller number of IL-8 after treatment with class B CpG ODN( Figure 5 C, Figure 5 D). These results suggest that IL-3 can act as an ADA2 blocker to reduce the expression of ADA2, thereby enhancing the activation of TLR9 by CpG ODNs. ADA2 regulates the activation of TLR9 in response to the binding of IL-3 to pDCs( Figure 8 ).

[0113] Furthermore, the present invention found that cells pretreated with a gene tool capable of reducing the expression of ADA2 (such as ADA2-specific siRNA) and activated by class B CpG 2006PTO secreted 8-fold and 2.5-fold more IFN-α and IL-8, respectively( Figure 7 A, Figure 7 B). When pDCs were treated with other classes of CpG ODNs, the secretion of IFN-α and IL-8 also increased significantly( Figure 7 C, Figure 7 D). These results suggest that a decrease in the expression of ADA2 in cells enhances the response of TLR9 to CpG ODNs PTO (i.e., a gene tool that reduces the expression of ADA2 can act as an ADA2 blocker).

[0114] Furthermore, the present invention found that ADA2 can bind to RNA, and the enzyme prefers RNA molecules with a linear structureFigure 3 E). In contrast, it has been reported that TLR9 does not bind to RNA. The present invention further shows that RNA that binds to ADA2 in endosomes may block the binding of CpG ODN to ADA2. In fact, pretreatment of pDCs with poly U before activation with class B CpG ODNs increased the secretion of IFN-α by 4-fold compared to activation with CpG ODN 2006PTO alone ( Figure 7 H). In contrast, transfection of pDCs with poly U did not induce significant release of IFN-α from the cells. Thus, the present invention proposes a new therapeutic strategy of blocking ADA2 with natural or chemically modified RNA (i.e., RNA can act as an ADA2 blocker) to activate pDCs in response to CpG ODNs.

[0115] The present invention has found that ADA2 competes with TLR9 for binding of CpG ODN in endosomes, thereby blocking the activation of TLR9 and the expression of IRF-7-dependent IFN-α and its secretion from pDCs ( Figure 8 Left). However, in lysosomes with a lower concentration of ADA2, these cells can still produce NF-κB-induced cytokines and chemokines (such as IL8) in response to the activation of TLR9 by class B CpG ODNs ( Figure 5 B, Figure 6 A).

[0116] In summary, the results of the present invention show that ADA2 plays a new role in pDC activation and the regulation of the adaptive immune system in response to innate immune responses. Co-activation of TLR9 with RNA and CpG ODNs and blocking of ADA2 can provide new therapeutic strategies to significantly induce the secretion of IFN-α from pDCs, thereby regulating the immune response to intracellular infections and cancer.

[0117] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit and scope protected by the present invention and the claims. These all fall within the protection scope of the present invention.

Claims

1. A composition comprising: (a) ADA2 blockers; and (b) TLR9 agonists; The ADA2 blocker comprises one or more of a gene tool for reducing ADA2 expression, a linear RNA and IL-3; the nucleotides in the linear RNA are connected by a phosphodiester bond or a phosphorothioate bond; and the TLR9 agonist is a CpG oligodeoxynucleotide.

2. The composition according to claim 1, characterized in that The linear RNA is a poly-U sequence.

3. The composition according to claim 1, characterized in that The TLR9 agonist is a class B CpG oligodeoxynucleotide, optionally including one or more of ODN 2006PTO, ODN BW006 PTO and ODN D-SL01PTO.

4. The composition according to claim 1, characterized in that The TLR9 agonist is a C-class CpG oligodeoxynucleotide, optionally including one or more of ODN D-SL03 PTO, ODN M362 PTO and ODN 2395 PTO.

5. The composition according to claim 1, characterized in that The genetic tool for reducing ADA2 expression includes ADA2-specific siRNA.

6. Use of the composition according to any one of claims 1 to 5 in the preparation of a medicament for treating intracellular infection and / or cancer.

7. The use according to claim 6, characterized in that The ADA2 blocker is administered prior to treatment with the TLR9 agonist.

8. The use according to claim 6, characterized in that The uses include enhancing the immune response against the intracellular infection and / or cancer.

9. The use according to claim 6, characterized in that The use includes increasing the secretion of IFN-α by plasmacytoid dendritic cells.

10. The use according to claim 6, characterized in that The intracellular infection optionally includes bacterial infection or viral infection; the cancer optionally includes solid tumors and hematological tumors.

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