A nucleic acid aptamer agonist conjugate and its preparation method and application

Abstract

The present invention provides a nucleic acid aptamer agonist conjugate and its preparation method and application. The nucleic acid aptamer agonist conjugate is prepared by coupling a nucleic acid aptamer, a linker and a STING agonist. The nucleic acid aptamer agonist conjugate can be used as a drug to target and identify targets on the surface of tumor cells and enter cells to activate the cGAS-STING signaling pathway to achieve an anti-tumor effect. The present invention also screens the types of STING agonists and linkers, so that the nucleic acid aptamer agonist conjugate not only has the advantages of easy transmembrane entry, strong tumor permeability, long half-life, and low toxic and side effects, but also has a strong inhibitory effect on triple-negative breast cancer. The nucleic acid aptamer agonist conjugate can also be combined with immune checkpoint blockade therapy to further achieve long-term and efficient inhibition of tumor growth.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a nucleic acid aptamer agonist conjugate and a preparation method and application thereof. Background Art

[0002] Innate immunity, the first line of defense in mammals against pathogen infection, activates the immune response by inducing the release of type I interferons and proinflammatory cytokines. The cyclic guanosine monophosphate (cGAS)-stimulator of interferon genes (STING) signaling pathway, a cytoplasmic innate immune pathway, plays a key role in antibacterial and antiviral responses, as well as in tumor immunotherapy. Activation of this pathway simultaneously induces both innate and adaptive immunity, playing a key regulatory role in tumor antigen release and presentation, T cell activation, and tumor infiltration, effectively reversing the immunosuppressive tumor microenvironment. STING agonists can also upregulate PD-L1 expression. Therefore, the cGAS-STING pathway, while exerting anti-tumor immunity, could also serve as a strategy to enhance the efficacy of immune checkpoint blockade therapy. However, due to the negatively charged phosphate groups of CDNs, they are difficult to translocate across the cell membrane, cannot be delivered systemically, have poor tumor penetration, and have a short half-life, resulting in poor clinical efficacy. On the other hand, when STING agonists act indiscriminately on normal tissues and tumor tissues, they can trigger toxic side effects such as systemic cytokine storms and lead to a narrow therapeutic window. Therefore, tumor-targeted delivery of CDNs-type STING agonists is an effective strategy to solve the above problems.

[0003] Aptamers are oligonucleotide sequences that bind specifically to targets through a defined three-dimensional structure. Their targets include small molecules, proteins, cells, tissues, and organs. Due to their high specificity and strong binding affinity, aptamers are also known as "chemical antibodies." Compared to antibodies, aptamers offer diverse screening methods, are easily synthesized and modified, have low immunogenicity, possess strong target affinity, and exhibit high tissue penetration. Therefore, they are widely used in targeted drug delivery. Aptamer-drug conjugates (ApDCs), constructed by conjugating aptamers to drug molecules, enable tumor-targeted drug delivery through the specific recognition of aptamers with tumor cell surface receptors, demonstrating excellent anti-tumor efficacy. Leveraging the tumor-targeting advantages of aptamers, they are expected to provide an effective solution for tumor-targeted delivery of STING agonists. Summary of the Invention

[0004] In response to the deficiencies of the prior art, the present invention provides a nucleic acid aptamer agonist conjugate, a preparation method thereof, and an application thereof. The nucleic acid aptamer agonist conjugate is prepared by coupling a nucleic acid aptamer, a linker, and a STING agonist. The nucleic acid aptamer agonist conjugate can be used as a drug to target and identify targets on the surface of tumor cells and activate the cGAS-STING signaling pathway to achieve an anti-tumor effect. The present invention also screens the types of STING agonists and linkers, so that the nucleic acid aptamer agonist conjugate not only has the advantages of easy transmembrane entry, strong tumor permeability, long half-life, and low toxic side effects, but also has a strong inhibitory effect on triple-negative breast cancer. The nucleic acid aptamer agonist conjugate combined with immune checkpoint blockade therapy can further achieve long-term and efficient inhibition of tumor growth.

[0005] In one aspect, the present invention provides a nucleic acid aptamer agonist conjugate, comprising a nucleic acid aptamer and a STING agonist, wherein the nucleic acid aptamer and the STING agonist are coupled together via a linker.

[0006] STING agonists can activate the interferon gene stimulator (STING) signaling pathway and the body's natural immune response. They are immunotherapy drugs suitable for a variety of cancers and produce sustained anti-tumor effects. However, STING agonists undergo passive and indiscriminate diffusion in the body, causing toxic side effects such as cytokine storms when acting on normal tissues, and can induce T cell apoptosis in a type I interferon-independent manner, thereby leading to inefficient target cell delivery. On the other hand, low-dose STING agonists can effectively inhibit tumor growth, but high doses can block CD8 ＋ The response of T cells promotes tumor growth, thus presenting a narrow therapeutic window. In particular, for cyclic dinucleotide STING agonists, due to the presence of their own negative charge, it is difficult for them to effectively enter the cell and have poor tumor penetration. Although intratumoral administration can improve the above problems to a certain extent, this mode of administration can only act on superficial tumors and has poor efficacy on metastatic tumors. Therefore, connecting nucleic acid aptamers to STING agonists containing linkers can provide an effective solution to the above problems, but different linkers will affect the release efficiency of STING agonists. The present invention constructs a conjugate of nucleic acid aptamers and STING agonists and selects suitable linkers to effectively solve the problems of STING agonists being unstable in the internal environment, having a short half-life, and not being easy to enter tumor cells across the membrane, and successfully achieves efficient targeted delivery of STING agonists.

[0007] Furthermore, the linker includes a cleavable linker and a non-cleavable linker; the cleavable linker includes any one or more of a GSH-responsive linker, a cathepsin-responsive linker, and an acid-responsive linker.

[0008] The conjugate of a cleavable linker and a drug. After the conjugate reaches the target site, the cleavable linker uses the difference between the tumor microenvironment and the normal physiological environment to release the drug, and the linker is cleaved through a specific chemical reaction in the tumor microenvironment.

[0009] The conjugate of the non-cleavable linker and the drug cannot be effectively broken down after the conjugate reaches the target site, resulting in the ineffective release of the STING agonist.

[0010] Previous research by the present invention team has found that aptamer-agonist conjugates coupled with non-cleavable linkers are unable to effectively release the STING agonist, making it difficult to activate the cGAS-STING signaling pathway. However, aptamer-agonist conjugates coupled with cleavable linkers exploit the differences between the tumor microenvironment and the normal physiological environment to disconnect the linker and the STING agonist, creating chemosensitive sites that can effectively release the STING agonist and achieve better tumor growth inhibition. Therefore, the use of cleavable linkers for conjugation is preferred. The aptamer-agonist conjugates prepared by the methods provided by the present invention have advantages such as strong tumor penetration, easy transcellular entry, long half-life, systemic administration, minimal toxicity and side effects, and a wide therapeutic window.

[0011] Furthermore, the cleavable linker includes any one or more of the following structural formulas:

[0012] 、 、 、 、 .

[0013] described 、 It is a GSH-responsive linker, which, under the action of high concentrations of GSH in tumor cells, undergoes a reduction reaction of the disulfide bond, resulting in the cleavage of the linker to release the STING agonist; 、 It is a cathepsin-responsive linker, which is cleaved and cleaved by cathepsins (such as cathepsin B) in tumor cells to release the STING agonist; It is an acid-responsive linker that uses the acidic internal environment of tumor cells to trigger the cleavage of the linker to release the STING agonist.

[0014] In some methods, different nucleic acid aptamer cyclic dinucleotide agonists are formed by coupling a cyclic dinucleotide agonist and Sgc8c with a cathepsin-responsive linker, a GSH-responsive linker, and an acid-responsive linker, respectively. The ability of the different nucleic acid aptamer cyclic dinucleotide agonists to induce SUM159 cells to produce CXCL-10 is then tested. The test results show that the nucleic acid aptamer cyclic dinucleotide agonist containing a cathepsin-responsive linker has a stronger ability to induce cells to produce CXCL-10, indicating that it has a stronger ability to activate the cGAS-STING signaling pathway.

[0015] Furthermore, the cathepsin-responsive linker is preferably

[0016] .

[0017] Furthermore, the STING agonist includes one or more of cyclic dinucleotides and non-cyclic dinucleotides.

[0018] Natural cyclic dinucleotide molecules have poor stability in the body and are easily degraded by nucleases in the body. Therefore, cyclic dinucleotide analogs are designed based on their structures. The cyclic dinucleotide analogs have the same functions as natural cyclic dinucleotides.

[0019] Furthermore, the structural formula of the cyclic dinucleotide is:

[0020] ;

[0021] wherein R1 or R1′ is one of H, F, -OH, and modified -OH; B1 is one of adenine or guanine; and B2 is one of adenine or guanine; Indicates that the phosphate bond can be connected to the 2' or 3' position of the pentose, and the sites that do not form a ring with the phosphate are replaced by R2 / R2', wherein R2 / R2' is one of H, F, -OH, and modified -OH; and R3 is one of O and S. The structural formula of the acyclic dinucleotide is:

[0022] .

[0023] After each substituent in the general structural formula of the cyclic dinucleotide is substituted, its stereoisomers, tautomers, stable isotope variants, pharmaceutically acceptable salts, prodrugs or solvates can all be used as cyclic dinucleotide STING agonists.

[0024] In some embodiments, the cyclic dinucleotide agonist is 3ʹ,3ʹ-di-(2ʹ-F)-cG s A s MP-2 (F2), the structural formula of F2 is:

[0025] .

[0026] The F2 is a compound in which the phosphate group is replaced by a thiophosphate. The thiophosphate structure can effectively resist degradation by a variety of extracellular and intracellular nucleases to increase the stability of F2 in the organism and prolong its half-life.

[0027] The acyclic dinucleotide The name of the drug is MSA-2. It exists in the form of monomer and dimer interconversion in the solution. Its dimer structure binds to the STING protein in tumor cells and can activate the cGAS-STING pathway.

[0028] In some methods, experiments have shown that the ability of the conjugate of the nucleic acid aptamer and the non-cyclic dinucleotide agonist MSA-2 to activate the cGAS-STING signaling pathway and the anti-tumor effect are not as good as those of free MSA-2, and the ability of the conjugate of the nucleic acid aptamer and the cyclic dinucleotide agonist F2 to activate the cGAS-STING signaling pathway and the anti-tumor effect are significantly better than those of free F2. Therefore, the STING agonist for preparing the nucleic acid aptamer agonist conjugate is preferably the cyclic dinucleotide agonist F2.

[0029] Furthermore, the nucleic acid aptamer agonist conjugate comprises any one or more of the following structural formulas:

[0030] 、

[0031] 、

[0032] 、

[0033] 、 、

[0034] 、 、

[0035] .

[0036] In some embodiments, the structural formula of the nucleic acid aptamer agonist conjugate is preferably:

[0037] .

[0038] Furthermore, the nucleic acid aptamer targets PTK7.

[0039] Theoretically, any nucleic acid aptamer can be used to prepare the nucleic acid aptamer agonist conjugate provided by the present invention. In order to improve the anti-tumor effect, nucleic acid aptamers that efficiently target tumor cells can be preferred to improve the targeted delivery effect of the agonist.

[0040] The nucleic acid aptamer is a DNA oligonucleotide sequence, and its 3′ end and / or 5′ end can be coupled with the linker and STING agonist provided by the present invention through modification.

[0041] In some embodiments, the nucleic acid aptamer is Sgc8c, and its target is PTK7. It was screened and optimized using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology, and its sequence is: ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA. Sgc8c can be modified with a 3′-thiol group or with both a 3′-thiol group and a 5′-amino group. These modifications are obtained by synthesizing on a DNA solid-phase synthesizer, followed by aminolysis, reduction, and HPLC purification. The 3′-thiol-modified Sgc8c can be linked to a single STING agonist, while the 3′-thiol- and 5′-amino-modified Sgc8c can be linked to two STING agonists.

[0042] The PTK7 is a cell surface transmembrane protein that is highly expressed in a variety of cancers, including triple-negative breast cancer, non-small cell lung cancer, ovarian cancer, colorectal cancer, gastric cancer, etc. Therefore, by recognizing PTK7 on the surface of the above-mentioned tumor cells through Sgc8c, the STING agonist is targeted and delivered to the tumor cells to exert its efficacy, which can minimize damage to normal cells, reduce side effects, and improve treatment effects.

[0043] In another aspect, the present invention provides a method for preparing a nucleic acid aptamer agonist conjugate, the method comprising the following steps:

[0044] (1) coupling a STING agonist to a linker; the STING agonist comprises one or more of a cyclic dinucleotide and a non-cyclic dinucleotide; the linker comprises a cleavable linker and a non-cleavable linker; the cleavable linker comprises any one or more of a GSH-responsive linker, a cathepsin-responsive linker, and an acid-responsive linker;

[0045] (2) Conjugating a STING agonist containing a linker to a nucleic acid aptamer.

[0046] In some embodiments, the molar ratio of the STING agonist containing a linker to the nucleic acid aptamer in step (2) is (3-5):1.

[0047] On the other hand, the present invention provides the use of F2 for preparing a reagent for improving the anti-tumor effect of nucleic acid aptamer agonist conjugates, wherein the structural formula of F2 is

[0048] .

[0049] In some methods, nucleic acid aptamer agonist conjugates prepared from the cyclic dinucleotide agonist F2 and nucleic acid aptamer agonist conjugates prepared from the non-cyclic dinucleotide agonist MSA-2 were tested for their inhibitory effects on triple-negative breast cancer. The test results showed that the nucleic acid aptamer agonist conjugate containing F2 had a significantly stronger inhibitory effect on triple-negative breast cancer than free F2, while the nucleic acid aptamer agonist conjugate containing MSA-2 had a significantly weaker inhibitory effect on triple-negative breast cancer than free MSA-2, indicating that preparing monomer F2 into a nucleic acid aptamer agonist conjugate can further improve the inhibitory effect of F2 on triple-negative breast cancer.

[0050] In another aspect, the present invention provides a composition for inhibiting tumor growth, comprising a nucleic acid aptamer agonist conjugate and a PD-1 monoclonal antibody, wherein the nucleic acid aptamer agonist conjugate is formed by coupling a nucleic acid aptamer and a STING agonist via a linker, wherein the STING agonist comprises one of a cyclic dinucleotide and a non-cyclic dinucleotide, and the linker comprises a cleavable linker and a non-cleavable linker; the cleavable linker comprises any one or more of a GSH-responsive linker, a cathepsin-responsive linker, and an acid-responsive linker.

[0051] In theory, the aptamer agonist conjugates provided by the present invention activate the cGAS-STING signaling pathway, promoting interferon production and immune cell activation, thereby reversing the immunosuppressive tumor microenvironment. Co-administration of these aptamer agonist conjugates with PD-1 monoclonal antibodies can further enhance the tumor-suppressing effect. PD-1 monoclonal antibodies inhibit tumor growth through immune checkpoint blockade therapy, based on the activation mechanism of immune cells called T cells. PD-1 (programmed death receptor 1) is expressed on the surface of T cells, while its ligand, PD-L1, is expressed on the surface of tumor cells. Binding of PD-1 to its ligand, PD-L1, can cause T cells to become exhausted and unable to kill tumor cells, allowing tumor cells to escape the host's immune surveillance. PD-1 monoclonal antibodies bind to PD-1 on the surface of T cells, blocking the binding of PD-L1 to PD-1, thereby maintaining T cell activation and preventing tumor cells from escaping immune surveillance. PD-1 monoclonal antibodies can enhance the killing effect of T cells on tumor cells, thereby exerting an anti-tumor effect.

[0052] In some embodiments, it is experimentally demonstrated that F2-L1-Sgc8c, having the structural formula

[0053] ,

[0054] The tumor inhibition rate of F2-L1-Sgc8c combined with PD-1 monoclonal antibody can not only be maintained at 70%-80% for a long time, but also allow mice to survive for a long time, indicating that F2-L1-Sgc8c activates the cGAS-STING signaling pathway and combines with immune blockade therapy to significantly improve the effect of tumor treatment. ＋ T / CD4 ＋ The T cell ratio proved that the combined administration of F2-L1-Sgc8c and PD-1 monoclonal antibody can significantly promote the maturation of DC cells in tumors and tumor-draining lymph nodes, and significantly increase the CD8 ＋ T / CD4 ＋ The proportion of T cells. Especially in tumors, the combined administration of F2-L1-Sgc8c and PD-1 monoclonal antibody showed an effect on increasing the CD8 ＋ Therefore, especially for the treatment of malignant tumor patients, the combined administration of F2-L1-Sgc8c and PD-1 provided by the present invention is expected to enhance the immune system of tumor patients and prolong their survival.

[0055] The present invention has the following beneficial effects:

[0056] (1) The present invention provides a nucleic acid aptamer agonist conjugate comprising a nucleic acid aptamer, a linker, and a STING agonist. The nucleic acid aptamer agonist conjugate can be used as a drug to target and recognize targets on the surface of tumor cells and activate the cGAS-STING signaling pathway to achieve an anti-tumor effect;

[0057] (2) The present invention also screened the types of STING agonists and linkers to make the nucleic acid aptamer agonist conjugates have stronger anti-tumor effects;

[0058] (3) The nucleic acid aptamer agonist conjugate not only has the advantages of easy transmembrane entry, strong tumor permeability, long half-life, and small toxic side effects, but also has a strong inhibitory effect on triple-negative breast cancer;

[0059] (4) The nucleic acid aptamer agonist conjugate can be used in combination with immune checkpoint blockade therapy to achieve long-term and high-efficiency inhibition of tumor growth and prolong survival. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 is the hydrogen spectrum of MSA-Mal;

[0061] Figure 2 is the mass spectrum of MSA-Mal-Sgc8c conjugate;

[0062] Figure 3 is the mass spectrum of MSA-NHS;

[0063] Figure 4 is the mass spectrum of Sgc8c-3′,5′-2MSA conjugate;

[0064] Figure 5 The expression of IFN-1 produced by THP1-dual-PTK7 reporter cells induced by nucleic acid aptamer STING agonist conjugates MSA-Mal-Sgc8c, Sgc8c-3′,5′-2MSA and monomer MSA-2;

[0065] Figure 6 Tumor volume, relative tumor volume, mouse body weight, and relative body weight changes of mice in in vivo anti-tumor experiments of the nucleic acid aptamer STING agonist conjugate MSA-Mal-Sgc8c and the monomer MSA-2;

[0066] Figure 7 is the hydrogen spectrum of compound 2;

[0067] Figure 8 is the hydrogen spectrum of linker L1;

[0068] Figure 9 is the mass spectrum of linker L1;

[0069] Figure 10 is the hydrogen spectrum of cyclic dinucleotide F2;

[0070] Figure 11 It is the high-resolution mass spectrum of cyclic dinucleotide F2;

[0071] Figure 12 is the mass spectrum of F2-L1;

[0072] Figure 13 is the mass spectrum of the F2-L1-Sgc8c conjugate;

[0073] Figure 14 The expression level of IFN-1 produced by THP1-dual-PTK7 reporter cells induced by the nucleic acid aptamer STING agonist conjugate F2-L1-Sgc8c and monomer F2;

[0074] Figure 15 Tumor volume, relative tumor volume, mouse body weight, and relative body weight changes of mice in the in vivo anti-tumor experiments of the nucleic acid aptamer STING agonist conjugate F2-L1-Sgc8c and monomer F2;

[0075] Figure 16 is the mass spectrum of F2-L2;

[0076] Figure 17 is the mass spectrum of the F2-L2-Sgc8c conjugate;

[0077] Figure 18 The expression levels of IFN-1 produced by THP1-dual-PTK7 reporter cells induced by F2-L1-Sgc8c coupled with a degradable linker and F2-L2-Sgc8c coupled with a non-degradable linker;

[0078] Figure 19 Tumor volume, relative tumor volume, mouse body weight, relative body weight changes, and survival curves of in vivo anti-tumor experiments using the nucleic acid aptamer STING agonist conjugate F2-L1-Sgc8c in combination with PD-1 monoclonal antibody, and F2-L1-Sgc8c and PD-1 alone;

[0079] Figure 20 This study is to study the in vivo anti-tumor mechanism after combined administration of F2-L1-Sgc8c and PD-1 monoclonal antibody, as well as after separate administration of F2, F2-L1-Sgc8c, and PD-1 monoclonal antibody. DETAILED DESCRIPTION

[0080] The present invention will be further described in detail below with reference to the accompanying drawings and examples. It should be noted that the following examples are intended to facilitate understanding of the present invention and do not have any limiting effect on the present invention.

[0081] Example 1: Effects of Aptamer-Acyclic Dinucleotide Agonist Conjugates on IFN-I Production and Tumor Inhibition

[0082] In this example, the acyclic dinucleotide agonist MSA-2 was conjugated to aptamers via different linkers to prepare aptamer-agonist conjugates to examine their effects on inducing IFN-I production and inhibiting tumors. Because PTK7 is widely expressed in various tumors, Sgc8c, a aptamer that specifically binds to PTK7 protein on the tumor cell surface and is internalized into the cell, was selected. Its sequence is: ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA. Sgc8c was selected and optimized using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method.

[0083] 1. Preparation of nucleic acid aptamer-acyclic dinucleotide agonist conjugates containing different linkers

[0084] (1) Preparation of MSA-Mal-Sgc8c

[0085] The acyclic dinucleotide agonist MSA-2 (50 mg, 0.17 mmol) and N-hydroxyethylmaleimide (96 mg, 0.68 mmol) were dissolved in 2 mL of anhydrous acetonitrile, and 10 μL of sulfuric acid was added. The reaction was carried out at 80 °C overnight. After the reaction, the solvent was evaporated and the target product MSA-Mal was separated by HPLC, a total of 35 mg. The hydrogen spectrum of MSA-Mal is as follows: Figure 1 The reaction formula is as follows:

[0086]

[0087] 3′-thiol-modified Sgc8c (4 μmol) was dissolved in 2.3 ml DPBS, and MSA-Mal (8.5 mg, 20 μmol) was dissolved in 1 ml acetonitrile. The two were mixed and reacted at 37 °C overnight. After the reaction was completed, 0.1 M TEAA was added to dilute the mixture. The target product MSA-Mal-Sgc8c was separated by HPLC. The mass spectrum of MSA-Mal-Sgc8c is shown in Figure 2. Figure 2 The reaction formula is as follows:

[0088]

[0089] (2) Preparation of MSA-NHS-Sgc8c

[0090] The non-cyclic dinucleotide agonist MSA-2 (50 mg, 0.17 mmol), N-hydroxysuccinimide (97 mg, 0.85 mmol) and TBTU (273 mg, 0.85 mmol) were dissolved in 2 ml of anhydrous dichloromethane, and then 30 μL of DIPEA was added. The reaction was carried out at 45 °C overnight. After the reaction, the mixture was washed with 5% citric acid solution, and the organic phase was collected. The solvent was evaporated and the mixture was redissolved in ethyl acetate. The precipitate was collected by centrifugation to obtain 32 mg of crude MSA-NHS. The mass spectrum of MSA-NHS is shown in Figure 2. Figure 3 The reaction formula is as follows:

[0091]

[0092] The 5′-amino-modified Sgc8c (1 μmol) was suspended in 1 ml of acetonitrile, and the MSA-NHS (2 mg, 5 mmol) prepared in (2) was added. The mixture was stirred at 37 °C overnight. After the reaction, the precipitate was collected by centrifugation, and the MSA-NHS-Sgc8c was obtained by ammonia cleavage and reduction, and then HPLC separation was performed. The reaction formula is as follows:

[0093]

[0094] (3) Preparation of Sgc8c-3′,5′-2MSA

[0095] 3′-Mercapto-Sgc8c-NHS-MSA (0.5 μmol) was dissolved in 400 μL 0.1 M TEAA, and MSA-Mal (1 mg, 2.5 μmol) was dissolved in 170 μL acetonitrile. The two were mixed and reacted at 37 °C overnight. After the reaction was completed, 0.1 M TEAA was added for dilution, and the target product Sgc8c-3′,5′-2MSA was obtained by HPLC separation. The mass spectrum of Sgc8c-3′,5′-2MSA is shown in FIG. Figure 4 The reaction formula is as follows:

[0096]

[0097] 2. Detect the ability of each nucleic acid aptamer STING agonist to induce cell production of IFN-Ⅰ

[0098] The four groups of additives shown in Table 1 below were added to THP1-dual-PTK7 reporter cells to examine the ability of each nucleic acid aptamer STING agonist to induce cells to produce IFN-I.

[0099] Table 1. Acyclic dinucleotide agonist conjugates with different linkers

[0100]

[0101] THP1-dual-PTK7 reporter cells are THP1-dual reporter cells that overexpress PTK7 and are used to determine the efficacy of STING agonists. The construction method is to construct a THP1-dual (THP1-dual-PTK7) reporter cell line that overexpresses PTK7 through lentiviral transfection. The specific steps are as follows: prepare 12 ml of 10 5 A THP1-dual cell suspension at 100 μg / mL was inoculated into a 6-well plate (2 mL / well). 60 μL of virus solution and 40 μL of infection reagent were added to each well. After centrifugation for 30 minutes, the cells were incubated at 37°C for 12 hours. The medium was then changed and cultured for an additional 72 hours. When the infection efficiency reached approximately 80% as observed under a microscope, puromycin (2 μg / mL) was added for resistance selection. After 48 hours of selection, the cells were replaced with the medium and puromycin (1 μg / mL) was added again. The infected cells were then screened and expanded.

[0102] The specific method is as follows: THP1-dual-PTK7 cells (10,000 / well) were plated in 96-well plates and cultured at 37°C and 5% CO2 for 18 hours. After that, MSA-2, MSA-Mal-Sgc8c, Sgc8c-3′, 5′-2MSA, and PBS were administered at a concentration of 20 μM. At 24 hours, 48 hours, 72 hours, and 96 hours, 10 μl of cell supernatant was transferred to a 96-well white microplate, 50 μl of QUANTI-Luc™ detection reagent was added, and the fluorescence intensity was detected using a microplate reader. PBS was used as a blank control, and the amount of IFN-I produced in the treatment group was expressed as a multiple of that in the PBS group. The results of the ability of each nucleic acid aptamer STING agonist to induce cells to produce IFN-I are shown in Tables 2 and 3. Figure 5 shown.

[0103] Table 2 Results of IFN-Ⅰ production induced by MSA-2 conjugates with different linkers

[0104]

[0105] According to Table 2 and Figure 5 The results showed that compared with free MSA-2, MSA-Mal-Sgc8c and Sgc8c-3′, 5′-2MSA showed a slow activation effect. The ability of MSA-Mal-Sgc8c and Sgc8c-3′, 5′-2MSA to induce cells to produce IFN-Ⅰ was weaker than that of free MSA-2, among which Sgc8c-3′, 5′-2MSA had the weakest ability to induce IFN-Ⅰ.

[0106] 3. Detection of the tumor inhibition ability of each nucleic acid aptamer STING agonist

[0107] 4T1 cells that highly express PTK7 protein were injected into the second mammary pad of 6-8 week-old female BALB / c mice. When the tumor grew to about 70-100 mm 3 The patients were randomly divided into three groups and injected with saline, MSA-2, and MSA-Mal-Sgc8c listed in Table 1, at a dose of 8.5 μmol / kg, once every three days for a total of three times. Tumor volume (V = 0.5 × L × W) was measured every three days. 2 ), calculate the average tumor volume of each group of mice, observe the difference in drug efficacy among the treatment groups, and when the tumor grows to 1500 mm 3 The mice were killed when the tumor of one mouse in each group grew to 1500 mm 3 The changes in tumor volume and relative tumor volume, weight and relative weight of mice in different dosing groups are shown in Figure 2. Figure 6The tumor volumes and inhibition rates of different drug administration groups are shown in Table 3 below, where the inhibition rate = (1-tumor volume of experimental group / tumor volume of Saline group) × 100%.

[0108] Table 3 Tumor inhibition rate of MSA-2 conjugates with different linkers

[0109]

[0110] According to Table 3 and Figure 6 Data analysis revealed no significant differences in weight changes between the groups of mice over the course of 0-14 days. In terms of anti-tumor efficacy, MSA-Mal-Sgc8c demonstrated comparable anti-tumor efficacy to free MSA-2. However, free MSA-2 has no targeted effect and, in addition to targeting tumor cells, also affects normal cells, potentially triggering a systemic cytokine storm, manifested by symptoms such as tissue damage and organ failure.

[0111] Example 2: Effects of Aptamer Cyclic Dinucleotide Agonist Conjugates on IFN-I Production and Tumor Inhibition

[0112] 1. Preparation of valine-alanine-maleimide linker

[0113] Compound 1 (1 g, 2.54 mmol) was dissolved in 30 ml of acetonitrile. The structural formula of compound 1 is:

[0114]

[0115] Then, a 4 M hydrochloric acid solution in dioxane (33 ml, 132 mmol) was added and the reaction was carried out at room temperature for 2 h. After the reaction was completed, the solvent was evaporated to dryness, the mixture was resuspended in acetonitrile, filtered, washed twice with ether, and the filter cake was collected to obtain compound 2. The hydrogen spectrum of compound 2 was as follows: Figure 7 As shown, the structural formula of compound 2 is:

[0116]

[0117] Without purification, it was directly carried out to the next step.

[0118] The crude compound 2 (780 mg, 2.5 mmol) was dissolved in 8 ml of DMF, and DIPEA (1.3 ml, 7.5 mmol) was added, followed by a DMF solution of 5-maleimidovalerate succinimidyl ester (882 mg, 3 mmol). The reaction was allowed to proceed at room temperature for 30 minutes. After completion of the reaction, the reaction solution was poured into a pre-cooled PBS solution. The precipitate was collected by centrifugation and washed with water, ether, and ethyl acetate, respectively, to obtain compound L1 (694 mg, 55% yield). The hydrogen spectrum of compound L1 is shown in FIG. Figure 8 As shown, the mass spectrum Figure 9As shown, the structural formula of compound L1 is:

[0119] .

[0120] The reaction formula for preparing the valine-alanine-maleimide linker L1 is as follows:

[0121]

[0122] 2. Preparation of STING agonists containing L1 linkers

[0123] The cyclic dinucleotide analog is 3ʹ,3ʹ-di-(2ʹ-F)-cG s A s MP isomer 3ʹ,3ʹ-di-(2ʹ-F)-cG s A s MP-2 is represented by F2, and the hydrogen spectrum of F2 is as follows Figure 10 As shown, the mass spectrum Figure 11 As shown, the structural formula of F2 is:

[0124]

[0125] Preparation of F2-L1: Under nitrogen protection, lyophilized F2 (10 mg, 10.9 μmol) was dissolved in 400 μL of ultra-dry DMF, followed by the addition of compound L1 (8.3 mg, 16.4 μmol) and sodium iodide (0.82 mg, 5.45 μmol). The reaction was carried out at 50 °C for 1 h. After the reaction, the product was directly purified by HPLC to obtain compound F2-L1 (3.5 mg, yield 27.5%). The mass spectrum of F2-L1 is shown below. Figure 12 shown.

[0126] The above reaction formula is as follows:

[0127]

[0128] 3. Preparation of nucleic acid aptamer STING agonist

[0129] The same nucleic acid aptamer Sgc8c as in Example 1 was selected.

[0130] 3′-thiol-modified Sgc8c (0.5 μmol) was dissolved in 500 μL DPBS, and the above-prepared F2-L1 (1.74 mg, 1.5 μmol) was dissolved in 500 μL acetonitrile. The two were mixed and reacted at 37 °C overnight. After the reaction was completed, 0.1 M TEAA was added to dilute the mixture, and the target product F2-L1-Sgc8c was obtained by HPLC separation. The mass spectrum of F2-L1-Sgc8c is shown in Figure 2. Figure 13As shown, the structural formula of F2-L1-Sgc8c is:

[0131] .

[0132] The above reaction formula is as follows:

[0133]

[0134] The ability of the F2-L1-Sgc8c conjugate prepared above to induce THP1-dual-PTK7 reporter cells to produce IFN-Ⅰ was tested. The F2-L1-Sgc8c group, the free F2 group, and the PBS group were set up. The detection method was referred to Example 1. The evaluation results are shown in Tables 4 and 5. Figure 14 As shown, the ability of cathepsin-responsive linker-coupled F2-L1-Sgc8c to induce IFN-I was significantly better than that of free F2.

[0135] Table 4 Results of IFN-Ⅰ production induced by F2-L1-Sgc8c and free F2 cells

[0136]

[0137] Furthermore, the ability of F2-L1-Sgc8c to inhibit 4T1 tumors that highly express PTK7 protein was tested. The F2-L1-Sgc8c group, the free F2 group, and the Saline group were set up. The detection method was similar to that in Example 1. The ability of the nucleic acid aptamer STING agonist to inhibit tumors was tested. The tumor volume and relative tumor volume changes, the weight changes, and relative weight changes of the mice in the different groups were as follows: Figure 15 The tumor volumes and inhibition rates of different drug administration groups are shown in Table 5 below.

[0138] Table 5. Tumor suppression effects of F2-L1-Sgc8c and free F2

[0139]

[0140] According to Table 5 and Figure 15Data analysis showed that there was no significant difference in the body weight of mice in each dosing group within 0-24 days. The tumor volume of the F2-L1-Sgc8c-dosing group was smaller than that of the free F2-dosing group at each time point, and the tumor inhibition rate was higher than that of the free F2-dosing group. F2-L1-Sgc8c showed a stronger tumor inhibition effect. Compared with the two, free F2 lacks targeting and acts on tumor cells and normal cells at the same time, which may trigger a systemic cytokine storm. In addition, F2 has poor tumor permeability, resulting in a relative decrease in free F2 enriched in tumor tissue. The F2-L1-Sgc8c conjugate exists stably in the body, targets tumor cells through nucleic acid aptamers, and has strong tumor permeability. Therefore, F2-L1-Sgc8c has a stronger tumor inhibition effect than free F2.

[0141] A comprehensive comparison of the experimental results of this example and the experimental results of Example 1 shows that the anti-tumor effect of F2-L1-Sgc8c is better than that of free MSA-2, and the cyclic dinucleotide agonist F2 is preferably used as the STING agonist for preparing nucleic acid aptamer agonist conjugates.

[0142] Example 3: Activation Effects of Aptamer Cyclic Dinucleotide Agonist Conjugates Containing Cleavable and Non-Cleavable Linkers on the STING Signaling Pathway

[0143] 1. Comparison of the activation effects of nucleic acid aptamer cyclic dinucleotide agonist conjugates prepared with cleavable and non-cleavable linkers on the STING signaling pathway

[0144] The cyclic dinucleotide agonist F2 was connected to different types of cleavable linkers and non-cleavable linkers to prepare nucleic acid aptamer agonist conjugates to examine their activation effects on the STING signaling pathway. The cleavable linker was L1 in Example 2, and its structural formula is , the non-cleavable linker is L2, and the structural formula is The nucleic acid aptamer selected was Sgc8c, which was the same as that in Example 1.

[0145] (1) Preparation of nucleic acid aptamer cyclic dinucleotide agonist conjugates containing cleavable and non-cleavable linkers

[0146] 1) Preparation of F2-L1-Sgc8c

[0147] F2-L1-Sgc8c was prepared according to the method provided in Example 2.

[0148] 2) Preparation of F2-L2-Sgc8c

[0149] Preparation of F2-L2: Under nitrogen protection, lyophilized and dehydrated F2 (5 mg, 5.4 μmol) was dissolved in 200 μL of ultra-dry DMF, followed by the addition of N-bromoethylmaleimide L2 (2.2 mg, 10.8 μmol) and sodium iodide (0.4 mg, 2.7 μmol). The reaction was carried out at 50 °C for 20 h. After the reaction, HPLC purification was performed directly to obtain compound F2-L2 (0.8 mg). The mass spectrum of F2-L2 is shown in FIG. Figure 16 As shown, the reaction formula is as follows:

[0150]

[0151] Preparation of F2-L2-Sgc8c: 3′-thiol-modified Sgc8c (0.5 μmol) was dissolved in 500 μL DPBS, and F2-L2 (1.25 mg, 1.5 μmol) was dissolved in 500 μL acetonitrile. The two were mixed and reacted at 37 °C overnight. After the reaction was completed, 0.1 M TEAA was added to dilute the mixture. The target product F2-L2-Sgc8c was separated by HPLC. The mass spectrum of F2-L2-Sgc8c is shown in Figure 2. Figure 17 shown.

[0152]

[0153] (2) Detecting the ability of nucleic acid aptamer cyclic dinucleotide agonist conjugates prepared with cleavable and non-cleavable linkers to induce cell production of IFN-Ⅰ

[0154] F2-L1-Sgc8c, F2-L2-Sgc8c, and PBS were added as additives to THP1-dual-PTK7 reporter cells to examine the ability of each nucleic acid aptamer STING agonist conjugate to induce cells to produce IFN-Ⅰ.

[0155] The specific steps are as follows: THP1-dual-PTK7 cells (50,000 / well) were plated in a 96-well plate and cultured at 37°C, 5% CO2 for 18 hours. F2-L1-Sgc8c, F2-L2-Sgc8c, and PBS supplements were administered at a concentration of 200 nM. After 24 hours of culture, 10 μl of cell supernatant was transferred to a 96-well white microplate plate, 50 μl of QUANTI-Luc™ detection reagent was added, and fluorescence intensity was measured using a microplate reader. PBS was used as a blank control, and the amount of IFN-1 produced in the treatment group was expressed as a multiple of that in the PBS group. The test results are shown in Tables 6 and 7 below. Figure 18 shown.

[0156] Table 6 Results of IFN-Ⅰ production induced by aptamer F2 conjugates containing lytic and non-lytic forms

[0157]

[0158] According to Table 6 and Figure 18 Data analysis showed that the ability of F2-L1-Sgc8c containing a cleavable linker to induce cell production of IFN-Ⅰ at a concentration of 200 nM was significantly higher than that of F2-L2-Sgc8c containing a non-cleavable linker, indicating that the nucleic acid aptamer agonist conjugate coupled with a cleavable linker has a better STING activation effect, while F2-L2-Sgc8c containing a non-cleavable linker cannot activate the STING pathway and produce IFN-Ⅰ due to the difficulty in effectively releasing F2. In addition, compared with the non-cleavable linker, the cleavable linker can release drugs under specific circumstances, and its drug release efficiency and accuracy are higher.

[0159] 2. Comparison of the activation effects of nucleic acid aptamer cyclic dinucleotide agonist conjugates prepared with different cleavage linkers on the STING signaling pathway

[0160] The above experiment 1 demonstrated that the nucleic acid aptamer cyclic dinucleotide agonist conjugate prepared with a cleavable linker had a stronger ability to induce IFN-1 production in THP1-dual-PTK7 cells. The following experiment will compare the STING activation effects of nucleic acid aptamer cyclic dinucleotide agonist conjugates prepared with different cleavable linkers.

[0161] The cleavable linkers include cathepsin-responsive linkers, GSH-responsive linkers, and acid-responsive linkers. The cathepsin-responsive linkers are L1 and L3, and their structural formulas are respectively

[0162] 、 ; The GSH response linkers are L4 and L5, and their structural formulas are 、 ; The acid-responsive linker is L6, and the structural formula is .

[0163] (1) Preparation of nucleic acid aptamer-cyclic dinucleotide agonist conjugates containing different cleavage-type linkers

[0164] 1) Preparation of F2-L1-Sgc8c

[0165] F2-L1-Sgc8c was prepared according to the method provided in Example 2.

[0166] 2) Preparation of F2-L3-Sgc8c

[0167] Preparation of linker L3: Under nitrogen protection, compound 3 (420 mg, 0.73 mmol) was dissolved in 5 ml of anhydrous DMF. A solution of thionyl chloride (104 mg, 0.87 mmol) in dichloromethane was slowly added dropwise in an ice bath and reacted for 30 min. The mixture was then transferred to room temperature and continued to react for 2 h. After vacuum concentration, linker L3 was obtained after column chromatography separation.

[0168] The above reaction formula is as follows:

[0169] .

[0170] Preparation of F2-L3: Under nitrogen, lyophilized F2 (10 mg, 10.9 μmol) was dissolved in 400 μL of ultra-dry DMF. Linker L3 (9.7 mg, 16.4 μmol) and sodium iodide (0.82 mg, 5.45 μmol) were then added. The reaction was incubated at 50 °C for 1 h and purified directly by HPLC to obtain compound F2-L3.

[0171] The above reaction formula is as follows:

[0172] .

[0173] Preparation of F2-L3-Sgc8c: 3′-thiol-modified Sgc8c (0.5 μmol) was dissolved in 500 μL DPBS, and F2-L3 (1.25 mg, 1.5 μmol) was dissolved in 500 μL acetonitrile. The two were mixed and reacted at 37 °C overnight. After the reaction was completed, 0.1 M TEAA was added to dilute the solution. The target product F2-L3-Sgc8c was obtained by HPLC separation.

[0174] 3) Preparation of F2-L4-Sgc8c

[0175] Preparation of Linker L4: 3-((2-Aminoethyl)disulfanyl)propionic acid (180 mg, 1 mmol) and maleic anhydride (186 mg, 2 mmol) were dissolved in 5 ml of a 3 / 1 acetic acid / toluene mixture and reacted at 120 °C overnight. After the reaction, the mixture was cooled to room temperature and then added with pentane in an ice bath. The precipitate was washed with ether and concentrated in vacuo to afford crude compound 6, which was carried on to the next step without purification. Under nitrogen, crude compound 6 (100 mg, 0.383 mmol) was dissolved in 5 ml of anhydrous dichloromethane and slowly added dropwise with thionyl chloride (91 mg, 0.766 mmol) in an ice bath. The reaction was continued in an ice bath for 30 min, then transferred to room temperature and allowed to react overnight. After the reaction, the mixture was concentrated in vacuo to afford crude linker L4, which was carried on to the next step without purification.

[0176] The above reaction formula is as follows:

[0177] .

[0178] Preparation of F2-L4: Under nitrogen protection, lyophilized F2 (10 mg, 10.9 μmol) was dissolved in 400 μL of ultra-dry pyridine, followed by the addition of linker L4 (15.2 mg, 54.5 μmol). The reaction was carried out at 50 °C for 40 h and directly purified by HPLC to obtain compound F2-L4.

[0179] The above reaction formula is as follows:

[0180] .

[0181] Preparation of F2-L4-Sgc8c: 3′-thiol-modified Sgc8c (0.5 μmol) was dissolved in 500 μL DPBS, and F2-L4 (1.25 mg, 1.5 μmol) was dissolved in 500 μL acetonitrile. The two were mixed and reacted at 37 °C overnight. After the reaction was completed, 0.1 M TEAA was added to dilute the solution. The target product F2-L4-Sgc8c was obtained by HPLC separation.

[0182] 4) Preparation of F2-L5-Sgc8c

[0183] Preparation of linker L5: Under nitrogen protection, L4 (100 mg, 0.36 mmol) was slowly added dropwise to a reaction system of paraformaldehyde (16.2 mg, 0.54 mmol) and zinc chloride (1 mg, 7.2 μmol) in an ice bath. The reaction was carried out in an ice bath for 30 min, and then the temperature was raised to 45 °C for 24 h. After the reaction, the filtrate was filtered, washed with DCM, and the filtrate was collected. The solvent was evaporated and separated by column chromatography to obtain linker L5.

[0184] The above reaction formula is as follows:

[0185] .

[0186] Preparation of F2-L5: Under nitrogen protection, lyophilized F2 (10 mg, 10.9 μmol) was dissolved in 400 μL of ultra-dry DMF. Linker L5 (4 mg, 13.1 μmol) and sodium iodide (0.82 mg, 5.45 μmol) were then added. The reaction was incubated at 50 °C overnight and directly purified by HPLC to obtain compound F2-L5.

[0187] The above reaction formula is as follows:

[0188] .

[0189] Preparation of F2-L5-Sgc8c: 3′-thiol-modified Sgc8c (0.5 μmol) was dissolved in 500 μL DPBS, and F2-L5 (1.25 mg, 1.5 μmol) was dissolved in 500 μL acetonitrile. The two were mixed and reacted at 37 °C overnight. After the reaction was completed, 0.1 M TEAA was added to dilute the solution. The target product F2-L5-Sgc8c was obtained by HPLC separation.

[0190] 5) Preparation of F2-L6-Sgc8c

[0191] Preparation of Linker L6: Dissolve the maleimide of compound 7 (1 g, 10 mmol) in 40 mL of ethyl acetate. Slowly add a solution of N-methylmorpholine (930 mg, 10.5 mmol) in ethyl acetate dropwise under an ice bath. Then, slowly add a solution of methyl chloroformate (1.23 g, 10 mmol) in ethyl acetate dropwise. After the addition is complete, transfer the mixture to room temperature and react for 1 h. After the reaction, filter the filtrate, dry it over anhydrous sodium sulfate, filter it, concentrate in vacuo, and recrystallize it from ethyl acetate and isopropyl ether to yield compound 9.

[0192] Compound 10 (n-propanolamine, 150 mg, 2 mmol) was dissolved in 6 ml of saturated sodium bicarbonate solution. Compound 9 (306 mg, 2 mmol) was added portionwise under ice-cooling. The mixture was allowed to react for 30 min under ice-cooling and then at room temperature for 20 min. After completion of the reaction, the mixture was extracted three times with chloroform. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated to yield compound 11.

[0193] Under nitrogen, compound 11 (116 mg, 0.5 mmol) was dissolved in 2 ml of anhydrous pyridine, and triphosgene (74 mg, 0.25 mmol) was dissolved in 2 ml of anhydrous dichloromethane. These were slowly added dropwise to compound 11 in an ice bath. After the addition, the mixture was moved to room temperature and reacted for 5 h. After completion of the reaction, the crude linker L6 was concentrated in vacuo to obtain the product. This was then carried out directly into the subsequent reaction without purification.

[0194] The above reaction formula is as follows:

[0195] .

[0196] Preparation of F2-L6: Under nitrogen protection, lyophilized F2 (10 mg, 10.9 μmol) was dissolved in 400 μL of ultra-dry pyridine, followed by the addition of linker L6 (16 mg, 55 μmol). The reaction was carried out at 50 °C for 40 h and directly purified by HPLC to obtain compound F2-L6.

[0197] The above reaction formula is as follows:

[0198] .

[0199] Preparation of F2-L6-Sgc8c: 3′-thiol-modified Sgc8c (0.5 μmol) was dissolved in 500 μL DPBS, and F2-L6 (1.25 mg, 1.5 μmol) was dissolved in 500 μL acetonitrile. The two were mixed and reacted at 37 °C overnight. After the reaction was completed, 0.1 M TEAA was added to dilute the solution. The target product F2-L6-Sgc8c was obtained by HPLC separation.

[0200] (2) Detection of the ability of nucleic acid aptamer F2 conjugates prepared with different cleavage-type linkers to induce cell production of CXCL-10

[0201] The six groups of additives shown in Table 7 below were added to SUM159 cells to examine the ability of each nucleic acid aptamer F2 conjugate to induce cells to produce CXCL-10.

[0202] Table 7. Aptamer F2 conjugates with different linkers

[0203]

[0204] The specific steps were as follows: SUM159 cells (5,000 / well) were plated in a 96-well plate and cultured at 37°C, 5% CO2 for 18 hours. The six supplements listed in Table 7 were then administered at a concentration of 100 nM. After 48 hours of culture, 100 μl of the cell supernatant was centrifuged and transferred to an ELISA kit. The absorbance at 450 nm was measured according to the manufacturer's instructions, and the CXCL-10 concentration was calculated. The results are shown in Table 8 below.

[0205] Table 8. Results of CXCL-10 production induced by aptamer F2 conjugates with different linkers

[0206]

[0207] According to the data analysis in Table 8 above, a comparison of the CXCL-10 production levels induced by SUM159 cells at a concentration of 100 nM using nucleic acid aptamer agonist conjugates coupled with different cleavable linkers showed that cathepsin-responsive linker-coupled F2-L1-Sgc8c and F2-L3-Sgc8c were significantly higher than the other groups, indicating that they can more effectively activate the cGAS-STING signaling pathway. The ability of the two cathepsin-responsive linker-coupled conjugates, F2-L1-Sgc8c and F2-L3-Sgc8c, to induce CXCL-10 production in SUM159 cells was compared, with F2-L1-Sgc8c being preferred.

[0208] In summary, L1 is preferably used as a linker for preparing nucleic acid aptamer cyclic dinucleotide agonists.

[0209] Example 4: Anti-tumor effect of aptamer agonist conjugate combined with PD-1

[0210] Example 3 experiments demonstrated that F2-L1-Sgc8c has a stronger ability to activate the cGAS-STING signaling pathway. This example explores the tumor treatment effect of F2-L1-Sgc8c activating the cGAS-STING signaling pathway combined with immune checkpoint blockade therapy.

[0211] The specific experimental method is: 4T1 cells that highly express PTK7 protein were injected into the second mammary pad of 6-8 week old female BALB / c mice. When the tumor grew to about 120 mm 3 The patients were randomly divided into five groups and injected with saline, F2-L1-Sgc8c conjugate (dose 0.11 μmol / kg), negative control F2-L1-40A conjugate (dose 0.11 μmol / kg, the 40A is a single-stranded DNA composed of 40 adenines), PD-1 monoclonal antibody (intraperitoneal administration, dose 5 mg / kg), F2-L1-Sgc8c conjugate & PD-1 monoclonal antibody (F2-L1-Sgc8c conjugate dose 0.11 μmol / kg, PD-1 monoclonal antibody intraperitoneal administration, dose 5 mg / kg) through the tail vein, once every three days for a total of five times. In the combined administration group, PD-1 monoclonal antibody was administered intraperitoneally the next day. Tumor volume (V = 0.5 × L × W) was measured every three days. 2 ), calculate the average tumor volume of each group of mice, observe the difference in drug efficacy among the treatment groups, and when the tumor grows to 1500 mm 3 The mice were killed when the tumor of one mouse in each group grew to 1500 mm 3 The changes in tumor volume and relative tumor volume, weight and relative weight of mice in different groups of mice treated with the drug, and the survival curves of mice are shown in Figure 2. Figure 19 The tumor volumes, inhibition rates and mouse survival rates of different dosing groups are shown in Tables 9 to 11 below.

[0212] Table 9. Tumor volume in different drug administration groups

[0213]

[0214] Table 10. Tumor inhibition rates in different drug groups

[0215]

[0216] Table 11. Survival rate of mice

[0217]

[0218] According to Table 9-Table 11 and Figure 19 Data analysis showed that compared with the negative control F2-L1-40A conjugate, the F2-L1-Sgc8c conjugate targeting the PTK7 protein showed a stronger tumor inhibitory effect, indicating that F2-L1-Sgc8c can effectively target tumor tissue after intravenous injection and significantly increase the enrichment of F2 in tumor tissue; at the same time, the combined administration of F2-L1-Sgc8c & PD-1 monoclonal antibody showed a stronger tumor inhibitory effect than the single administration of F2-L1-Sgc8c conjugate or PD-1 monoclonal antibody, significantly prolonging the survival period of tumor-bearing mice, with a survival rate of 100% on the 40th day, and ultimately 20% of the mice achieved long-term survival. In contrast, the tumors of all mice in the other groups had grown to 1500 mm on the 40th day. 3 This indicates that the combined administration of nucleic acid aptamer cyclic dinucleotide agonist conjugate F2-L1-Sgc8c and PD-1 monoclonal antibody not only has a stronger tumor inhibitory effect, but also can significantly prolong the survival of tumor-bearing mice.

[0219] It should be noted that the experiments on inhibiting mouse tumors in Example 2 and the experiments on inhibiting mouse tumors in this example are not the same batch of experiments, and the starting volumes of the inhibited tumors are different, so there are differences in the tumor inhibition effects.

[0220] To further explore the mechanism of tumor growth inhibition by F2-L1-Sgc8c and F2-L1-Sgc8c & PD-1 monoclonal antibody combination, the DC cell maturation ratio and CD8 ＋ T / CD4 ＋ T cell ratio. Test results are as follows Figure 20 As shown in the results, compared with free F2, the F2-L1-Sgc8c conjugate can significantly promote the maturation of DC cells in tumors and tumor-draining lymph nodes, and significantly increase the expression of CD8 ＋ T / CD4 ＋ The proportion of T cells showed more CD8 ＋ T cells are recruited to the tumor site to play an anti-tumor role. When the F2-L1-Sgc8c conjugate is combined with PD-1 monoclonal antibody, the DC cell maturation ratio and CD8 ＋ T / CD4 ＋ The proportion of T cells was further increased, especially in tumors. The combined administration of F2-L1-Sgc8c and PD-1 monoclonal antibody showed an effect on increasing CD8 ＋ T / CD4 ＋The proportion of T cells has a synergistic enhancing effect, indicating that the F2-L1-Sgc8c conjugate can effectively reverse the immunosuppressive tumor microenvironment, thereby improving the anti-tumor efficacy of immune checkpoint inhibitors.

[0221] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the scope defined by the claims.

Claims

1. A nucleic acid aptamer agonist conjugate, characterized in that: The invention comprises a nucleic acid aptamer and a STING agonist, wherein the nucleic acid aptamer and the STING agonist are coupled to form a whole through a linker; the nucleic acid aptamer is Sgc8c; the STING agonist comprises one or more of cyclic dinucleotides and non-cyclic dinucleotides, and the structural formula of the cyclic dinucleotide is , the structural formula of the acyclic dinucleotide is ; The linker is a cleavable linker, and the cleavable linker includes any one or more of the following structural formulas: 、 、 、 、 。 2. The nucleic acid aptamer agonist conjugate according to claim 1, wherein The nucleic acid aptamer agonist conjugate includes any one or more of the following structural formulas: 、 、 、 、 。 3. The method for preparing the nucleic acid aptamer agonist conjugate according to any one of claims 1 to 2, wherein: The following steps are involved: (1) Conjugating the STING agonist to the linker; (2) Conjugating a STING agonist containing a linker to a nucleic acid aptamer.

4. Use of the nucleic acid aptamer agonist conjugate according to claim 1 in the preparation of an anti-triple-negative breast cancer drug.

5. A composition for inhibiting tumor growth, characterized in that: It includes a nucleic acid aptamer agonist conjugate and a PD-1 monoclonal antibody, wherein the nucleic acid aptamer agonist conjugate is formed by coupling a nucleic acid aptamer and a STING agonist via a linker; the nucleic acid aptamer is Sgc8c, and the STING agonist has the structural formula , the structural formula of the linker is .

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