Anti-tumor deoxyoligonucleotide containing CpG motif and interfering PD-L1 expression

Abstract

The invention provides a CpG motif-containing anti-tumor deoxyoligonucleotide for interfering PD-L1 expression, which is named as ILO, and the sequence of the anti-tumor deoxyoligonucleotide is as shown in a sequence table lt; 400 gt; 1 is shown in the specification. The ILO can activate a Toll-like receptor 9 (TLR-9) and inhibit the expression of PD-L1 in tumor cells and T lymphocytes, so that the anti-tumor innate immunity and adaptive immune response of a body are enhanced at the same time. The ILO can effectively inhibit tumor growth and prolong the survival time of tumor-bearing individuals. The oligonucleotide not only can be independently used as an anti-tumor drug, but also can be combined with other anti-tumor drugs, and is suitable for treating various tumors including lung cancer and melanoma. In addition, ILO can also be used as a preparation for enhancing the efficacy of a tumor vaccine or an influenza vaccine.

Description

Technical Field

[0001] This invention relates to an antitumor deoxygenated oligonucleotide containing a CpG motif that interferes with PD-L1 expression. This oligonucleotide has a specific sequence that activates Toll-like receptor 9 (TLR-9) and inhibits PD-L1 expression. This oligonucleotide can be used alone or in combination with other antitumor agents to treat tumors, and can also be used as an adjuvant for tumor vaccines and to enhance the efficacy of influenza virus vaccines. Background Technology

[0002] Programmed death-ligand 1 (PD-L1) is a membrane protein expressed on cancer cells in humans and rodents [Blood. 2008 Apr 1;111(7):3635-43]. PD-L1 expressed on cancer cells can interact with programmed cell death protein 1 (PD1) on T cells [EMBO J. 1992 Nov;11(11):3887-95; Immunity. 2016 May 17;44(5):1052-68], thereby initiating an inhibitory signaling pathway for T cell activation. This inhibitory signal suppresses T cell function [Honda et al., Immunity. 2014 Feb 20;40(2):235-247]. In the tumor microenvironment, tumor cells suppress the anti-tumor function of T cells by expressing PD-L1. Due to its T-cell-suppressing function, PD-L1 is an immune checkpoint (IC) protein [Eur J Clin Pharmacol. 2026 Jan 17;82(2):35]. Immune checkpoint proteins act as "brakes" on the immune system. These "brakes" can be released by immune checkpoint inhibitors (ICIs). ICIs are a class of agents that maintain and promote T-cell activation by blocking, inhibiting, or weakening inhibitory signals that mediate immune checkpoint-mediated T-cell activation. ICIs can "release the brakes," reactivating and enhancing the ability of T cells to recognize and kill tumors, thereby producing a tumor therapeutic effect. Anti-PD-L1 antibodies can block the binding of PD-L1 and PD-1, thereby reducing the inhibitory signals of T cell activation, thus restoring and enhancing the anti-tumor activity of T cells [Cell Rep. 2017 Aug 22;20(8):1818-1829;Lancet. 2017 Jan 21;389(10066):255-265].Atezolizumab [Lancet. 2017 Jan 21;389(10066):255-265], durvalumab [N Engl J Med. 2017 Nov 16;377(20):1919-1929], and avelumab [Lancet Oncol. 2016 Oct;17(10):1374-1385] are three FDA-approved humanized antibodies (anti-PD-L1 antibodies) targeting PD-L1, used to treat a variety of malignancies, including non-small cell lung cancer, urothelial carcinoma, and triple-negative breast cancer. Atezolizumab prolongs overall survival in patients with non-small cell lung cancer [Lancet. 2017 Jan 21;389(10066):255-265; Lancet. 2019 May 4;393(10183):1819-1830]. Chemotherapy delay: The standard chemotherapy regimen of pembrolizumab combined with pemetrexed and platinum drugs significantly prolongs overall survival and progression-free survival in patients with non-small cell lung cancer (NSCLC) compared with chemotherapy alone [N Engl JMed. 2018 May 31;378(22):2078-2092].

[0003] PD-L1 is also expressed on the surface of dendritic cells [J Immunol. 2003 Feb 1;170(3):1257-66] and activated T cells [J Immunol. 2003 Feb 1;170(3):1257-66]. Inhibition of PD-L1 on the surface of dendritic cells and activated T cells can also produce anti-tumor effects because these two types of cells play key roles in the immune response. PD-L1 binding to PD-1 on T cells transmits inhibitory signals, weakening T cell function. Blocking the PD-L1 on these immune cells themselves can relieve their direct inhibitory effect on effector T cells, maintaining T cell activation and killing ability. At the same time, reducing PD-L1 expression on dendritic cells can also enhance their antigen presentation and co-stimulatory functions, thereby more effectively initiating a strong anti-tumor immune response.

[0004] Anti-PD-L1 can induce immune-related adverse events (irAEs) through complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). The occurrence of these irAEs is because PD-L1 is also expressed in macrophages [Proc Natl Acad Sci US A. 2003 Apr 29;100(9):5336-41], endothelial cells [Eur J Immunol. 2003 Nov;33(11):3117-26;Microcirculation. 2002 Apr;9(2):133-45;J Immunol. 2002 Oct 1;169(7):3581-8], corneal epithelial cells [J Immunol. 2007 Sep 15;179(6):3672-9], placental trophoblast cells [Biol Reprod. 2003 May;68(5):1496-504], and dendritic cells [J Immunol. 2003 Feb 15]. The surface of the normal cells, including pancreatic islet cells [Diabetologia.2025 Feb;68(2):382-396], activated T cells [J Immunol. 2003 Feb 1;170(3):1257-66], placental trophoblast cells [J Immunol. 2003 Feb 1;170(3):1257-66], myocardial endothelial cells [J Immunol.2003 Feb 1;170(3):1257-66], and thymic cortical epithelial cells [J Immunol. 2003 Feb 1;170(3):1257-66]. Anti-PD-L1 antibodies recognize and bind to PD-L1 on these normal cells, which initiates antibody-mediated CDC and ADCC, thereby causing immune damage to tissue cells and leading to irAEs in individuals.Irritable bowel syndrome (irAEs) can manifest as pneumonia, hepatitis, myocarditis, arrhythmia, uveitis, hypopituitarism, endocrine disorders, neuropathy [JAMA Oncol.2018 Dec 1;4(12):1721-1728; N Engl J Med. 2018 Jan 11;378(2):158-168; Eur J Clin Pharmacol. 2026 Jan 17;82(2):35], Guillain-Barré syndrome, myasthenia gravis, colitis, hepatitis, arthritis, immune-related skin diseases, and endocrine disorders (diabetes, adrenal insufficiency, and hypopituitarism) [Eur J Clin Pharmacol. 2026 Jan 17;82(2):35]. This antibody-mediated immune damage mechanism to normal cells reveals a key limitation of using anti-PD-L1 antibodies to treat tumors, and highlights the necessity of developing non-antibody PD-L1 inhibitors that can effectively reduce PD-L1-induced T-cell activation inhibitory signals while minimizing irAEs.

[0005] In recent years, with the continuous deepening of research, small nucleic acid drugs have shown increasingly broad clinical application prospects. Currently, 11 antisense oligonucleotides (ASO), 2 aptamers and 6 siRNAs have been approved as small nucleic acid drugs for clinical use [Signal TransductTarget Ther. 2025 Mar 10;10(1):73].

[0006] Although anti-PD-L1 antibodies have shown efficacy in the treatment of various tumors, their effectiveness is still unsatisfactory, with a treatment rate of only 10%-30% for most malignant tumors [Ribas, al., Science 359, no. 6382 (2018):1350-1355]. Therefore, it is necessary to develop more effective immune checkpoint inhibitors targeting PD-L1 for tumor treatment. Summary of the Invention

[0007] 1. This invention provides an antitumor deoxy oligonucleotide containing a CpG motif that interferes with PD-L1 expression, the sequence of which is shown in the sequence listing. <400> As shown in Figure 1, this deoxyoligonucleotide was named ILO.

[0008] 2. ILO can activate Toll-like receptor 9 (TLR-9) and inhibit PD-L1 expression. 3. ILO can enhance an individual's innate and adaptive anti-tumor immune responses. 4. After application to individuals, ILO can inhibit tumor growth and prolong the survival of tumor-bearing individuals. 5. ILO can be used alone or in combination with other anti-tumor drugs for tumor treatment. 6. ILO can enhance the efficacy of influenza virus vaccines.

[0009] Terminology in the invention:

[0010] Unless otherwise specified, the terminology used in this invention has the ordinary meaning that can be understood by those skilled in the art. In the event of any conflict of meaning, the interpretations, definitions, or descriptions in this invention shall prevail.

[0011] Oligonucleotides are molecules composed of several to dozens of nucleotides. Nucleotides are the basic structural units of nucleic acids and oligonucleotides, consisting of a nucleoside and a phosphate group. A nucleoside is composed of a pentose sugar (including ribose and deoxyribose) and a base. The pentose sugar and base are linked to form a nucleoside, which is then linked by a phosphate group to form a nucleotide. Nucleotides are linked by phosphodiester bonds to form oligonucleotides. The bases in nucleosides include pyrimidines and purines. The main pyrimidine bases are thymine (T or t) and cytosine (C or c); the main purine bases are adenine (A or a) and guanine (G or g). In addition, oligonucleotides may also contain rare bases, such as 5-hydroxymethylcytosine and 7-methylguanine. Oligonucleotides can be single-stranded, double-stranded, cyclic, or other cyclic structures. In this invention, the abbreviation "ODN" may be used to refer to oligonucleotides. The primary structure of an oligonucleotide is determined by the sequence of its nucleotides, also known as the nucleotide sequence. Since the nucleotide sequence can be represented by a base sequence, it is often referred to as the base sequence. For deoxy oligonucleotides, their sequence can be represented using the abbreviations of bases, such as T / t for thymine, C / c for cytosine, A / a for adenine, and G / g for guanine.

[0012] "Oligonucleotide provided by this invention": The oligonucleotide provided by this invention is an antitumor deoxygenated oligonucleotide containing a CpG motif that interferes with PD-L1 expression, and its sequence is shown in the sequence listing. <400> As shown in Figure 1, it is named ILO. ILO possesses a unique and novel primary structure containing a CpG motif, thus enabling it to activate the innate immune response. ILO also has the function of interfering with PD-L1 mRNA, thereby inhibiting PD-L1 protein expression. Therefore, ILO is a bifunctional immunomodulatory oligonucleotide.

[0013] "Chemical Modification": Compared to natural DNA, the antitumor deoxy oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided by this invention can be chemically modified. Chemical modification refers to the method of altering its covalent structure by introducing or removing chemical groups. Chemical modification sites for ILO can occur at the phosphodiester bond, ribose, and bases, as well as at the 5' or 3' end. Chemical modification can be performed during or after synthesis. The types of modification covered by this invention include, but are not limited to: oligonucleotide backbone modification (such as thiomodification, alkyl / aromatic substitution, etc.); base substitution (using rare bases or derivatives) and base modification; and the attachment of one or more nucleotides or other chemical groups at the 5' and / or 3' ends.

[0014] "Immune Response": The anti-tumor deoxygenated oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression, provided by this invention, can be used for individual cancer treatment by enhancing the immune response to tumor cells and tumor antigens. Immune response has the same meaning as immune reaction. Immune response is divided into innate immune response and adaptive immune response. An immune response against tumor cells can limit tumor cell growth and eliminate tumor cells. Promoting an individual's immune response to tumor cells produces an anti-tumor effect, thus prolonging the survival of cancer patients.

[0015] "Individual": In this invention, "individual" refers to humans and non-human vertebrates.

[0016] "Tumor": In this invention, the terms "tumor" and "cancer" are used interchangeably. A tumor is a general term for a class of diseases, including solid tumors, soft tissue tumors, and hematologic malignancies originating from the myeloid or lymphoid systems. Common solid tumors include lung cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer, liver cancer, and melanoma; soft tissue tumors encompass types such as liposarcoma, leiomyosarcoma, and synovial sarcoma; while myeloid and lymphoid system tumors mainly include various types of leukemia (such as acute myeloid leukemia and chronic lymphocytic leukemia), lymphomas (such as Hodgkin's lymphoma and diffuse large B-cell lymphoma), and multiple myeloma. The antitumor deoxyribonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided by this invention can be used alone or in combination with other antitumor agents to treat tumors. These tumors include, but are not limited to, lung cancer and melanoma.

[0017] "Tumor treatment": Tumor treatment refers to the individual application of anti-tumor agents to control tumor progression, prolong survival, improve quality of life, alleviate symptoms, shrink or even eliminate tumors, or inhibit tumor metastasis. In this invention, "tumor treatment" and "anti-tumor effect" or "treatment of tumors" have the same meaning. Anti-tumor effect also includes the prevention of tumor occurrence, recurrence, and metastasis. The anti-tumor deoxyoligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided by this invention can be used for tumor treatment.

[0018] "Anti-tumor agent": The "anti-tumor agent" as described in this invention refers to a pharmaceutical composition or biological agent that, through direct or indirect mechanisms of action, can produce a tumor therapeutic effect after being administered to an individual. In this patent document, the terms "preparation" and "drug" are equivalent and can be used interchangeably; "anti-tumor agent" and "anti-tumor drug" are also equivalent concepts and can be used interchangeably in the specification and claims. The anti-tumor agent includes, but is not limited to, the following categories: cytotoxic chemotherapy drugs; tumor vaccines; immune checkpoint inhibitor antibodies; oncolytic viruses; angiogenesis inhibitors; tumor therapeutic antibodies; small molecule kinase inhibitors; cell therapy agents, including but not limited to dendritic cells, chimeric antigen receptor T cells (CAR-T), tumor-infiltrating lymphocytes, and natural killer cells. Those skilled in the art should understand that the above classification is merely illustrative and is not a limitation on the scope of protection. Any existing or future-developed drug or biological agent with anti-tumor activity, as long as it can be used alone or in combination with the anti-tumor deoxyribonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression as described in this invention, falls within the definition of "anti-tumor agent" in this patent.

[0019] "Anti-tumor T-cell immune response": The anti-tumor T-cell immune response is an anti-tumor immune response mediated by T lymphocytes. T cells (T lymphocytes) are the most important anti-tumor effector cells in the adaptive immune system, mainly including CD8⁺ T cells and CD4⁺ T cells. CD8⁺ T cells are cytotoxic T lymphocytes (CTLs) that express CD8 molecules on their surface, and can directly recognize and kill tumor cells; CD4⁺ T cells are helper T lymphocytes (Th) that express CD4 molecules on their surface, and in addition to assisting CD8⁺ T cells in their killing function, they can also directly kill tumor cells or mediate tumor cell death by secreting cytokines (such as tumor necrosis factor) [Cao LL, et al. Immunity. 2023 Oct 10;56(10):2206-2217]. The anti-tumor deoxygenated oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided in this invention can exert an anti-tumor effect by promoting and maintaining the activation state of anti-tumor T cells.

[0020] "Tumor microenvironment": The tumor microenvironment (TME) refers to a complex and dynamic ecosystem surrounding tumor cells. It includes not only the tumor cells themselves, but also infiltrating immune cells (such as T cells and macrophages), fibroblasts, vascular endothelial cells, and non-cellular components such as extracellular matrix, signaling molecules, and metabolites. These components interact with each other to shape the biological characteristics of the tumor and profoundly affect its growth, invasion, metastasis, and response to treatment [Mol Med Rep. 2026 Mar;33(3):93; J Cancer. 2019 Aug 7;10(19):4574-4587].

[0021] "Cold tumor microenvironment": A cold tumor microenvironment (Cold TME) is a tumor region lacking infiltration of immune cells, especially T cells. It often contains a large number of regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and inhibitory cytokines. Tumor cells in a cold tumor microenvironment can easily escape the attack of immune cells, especially T cells. This makes the tumor respond poorly to immunotherapy. Tumors with a cold tumor microenvironment can be called cold tumors [Mol Med Rep. 2026 Mar;33(3):93].

[0022] "Hot tumor microenvironment": The hot tumor microenvironment (Hot TME) refers to a tumor region rich in immune cell infiltration and highly immunogenic. Its main characteristics are a high density of tumor-infiltrating lymphocytes, especially activated cytotoxic T cells. This environment typically exhibits an active immune response, including elevated levels of inflammatory cytokines such as interferon-γ, enhanced antigen-presenting function, and relatively few immunosuppressive cells. Tumor cells in the hot tumor microenvironment are more susceptible to attack by immune cells, especially T cells. Transforming the tumor from an immunologically "cold" state to a "hot" state is a promising strategy for enhancing the efficacy of immunotherapy. Tumors with a hot tumor microenvironment can be termed hot tumors [Mol Med Rep. 2026 Mar;33(3):93]. The antitumor deoxyribonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided in this invention can induce a hot tumor microenvironment.

[0023] "Tumor-infiltrating lymphocytes": Tumor-infiltrating lymphocytes (TILs) refer to a heterogeneous population of lymphocytes that migrate into the tumor microenvironment, mainly including T cells, B cells, and NK cells, with T cells being the predominant group [Oncol Rep. 2026 Mar;55(3):42]. TILs interact dynamically with cancer cells and participate in the immunoediting process of cancer [J Intern Med. 2016;279:541–562]. This process includes three phases: elimination, equilibrium, and escape [Curr Opin Immunol. 2014;27:16–25]. The elimination phase occurs when tumor antigens are bound to human leukocyte antigen molecules by antigen-presenting cells and presented to CD8. + Cytotoxic T lymphocytes, when recognized by T cell receptors [Nat RevCancer. 2012;12:265–277]. Most TILs are T cells, including CD4+. + T lymphocytes and CD8 + T lymphocytes. CD4 + T lymphocytes initiate tumor-specific CD8 activation. + TILs are crucial [Nature. 2003;421:852–856]. During equilibrium, FOXP3 protein inhibits CD4... + The generation of regulatory T cells (Tregs) plays a crucial role; these cells promote immunosuppression, thereby inducing resistance to CD8+. + T-cell immune tolerance. FOXP3 is also involved in immune escape mechanisms because it can reduce the immune response in the tumor microenvironment [Science. 2003;299:1057–1061]. During equilibrium, due to decreased FOXP3 protein expression, the tumor microenvironment exhibits a high proportion of cytotoxic T cells and a low proportion of Tregs, which promotes tumor progression to the next stage [Nat Rev Immunol. 2010;10:490–500]. The antitumor deoxyribonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided in this invention can significantly increase the number of CD4⁺ T lymphocytes and CD8⁺ T lymphocytes in tumor-infiltrating lymphocytes and inhibit the expression of PD-L1 in these two cell types.

[0024] "Regulatory T lymphocytes": Regulatory T cells (Tregs) are a key subset of suppressive CD4⁺ T cells in the immune system, whose development and function are primarily governed by the transcription factor FOXP3. These cells actively suppress the activation and function of immune cells such as effector T cells and antigen-presenting cells through various mechanisms, including secreting suppressive cytokines, consuming interleukin-2, and expressing suppressive receptors, playing a core role in negative immune regulation in the tumor microenvironment [Curr Opin Immunol. 2007;19:217–223]. During the equilibrium period of immune editing, FOXP3 protein plays a crucial role in the generation of CD4⁺ regulatory T cells [Cell. 2026;189(1):6-22]. These cells promote immunosuppression, leading to immune tolerance to CD8⁺ T cells. Overexpression of FOXP3 is associated with regulatory T cell proliferation and severe immunodeficiency, while its absence leads to immune system activation. FOXP3 is also involved in immune escape mechanisms and affects the low survival rate of breast cancer patients because it works by reducing the immune response within the tumor [Science. 2003;299:1057–1061]. During equilibrium, due to the reduction of FOXP3 protein, the tumor microenvironment exhibits a high proportion of cytotoxic T cells and a low proportion of regulatory T cells, allowing the tumor to progress to the next stage [Nat Rev Immunol. 2010;10:490–500]. The antitumor deoxyribonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided in this invention can significantly reduce the number of Tregs in the tumor microenvironment.

[0025] Pattern recognition receptors (PRRs) are a class of protein molecules expressed on the surface or intracellularly of innate immune cells and some adaptive immune cells. Their main function is to recognize highly conserved molecular patterns in pathogens or danger signals, namely pathogen-associated molecular patterns and damage-associated molecular patterns. This family includes Toll-like receptors, RIG-I-like receptors, NOD-like receptors, and C-type lectin receptors [Cao LL, et al. Immunity. 2023;56(10):2206-2217]. When a pattern recognition receptor binds to its ligand, it can initiate downstream signal transduction pathways, such as activating NF-κB or interferon regulators, thereby inducing the production of pro-inflammatory cytokines, chemokines, and type I interferons. In the context of tumor immunity, B cells and dendritic cells can sense relevant signals in the tumor microenvironment through pattern recognition receptors, upregulate the expression of co-stimulatory molecules, and thus enhance their ability to present tumor antigens, promoting the activation of anti-tumor T cells to kill tumor cells.

[0026] "Toll-like receptor 9 agonists": Toll-like receptor 9 (TLR9) is a pattern recognition receptor primarily expressed in immune cells such as B cells and plasmacytoid dendritic cells. Deoxyribonucleotides containing CpG motifs (CpG ODNs) can mimic the unmethylated CpG motifs in bacterial or viral DNA, thereby effectively activating the Toll-like receptor 9 signaling pathway. CpG-containing deoxy oligonucleotides (CpG ODNs) can shape the antitumor immune microenvironment [Hemmi H, et al. Nature 408, 740–745 (2000); Celhar T, et al., Int. Immunol 28, 223–232 (2016); Schnare M, et al. Curr. Biol 10, 1139–1142 (2000); Khodadoust MS, et al. Blood 133, 878–881 (2019); Browne EP. Immunology. 2012 Aug;136(4):370-9]. CpG-containing deoxygenated oligonucleotides can activate B cells and dendritic cells, upregulating co-stimulatory molecules including CD86, making them more efficient antigen-presenting cells that provide a second activation signal to anti-tumor T cells [Cao LL, et al. Immunity. 2023 Oct 10;56(10):2206-2217;Sagiv-Barfi I, et al. Sci Immunol.2022 May 27;7(71):eabn5859]. CpG-containing deoxygenated oligonucleotides activate B cells and upregulate their co-stimulatory molecules through Toll-like receptor 9, thereby activating anti-tumor T cells to kill tumor cells, thus having a tumor therapeutic effect. The anti-tumor deoxygenated oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided in this invention has been sequence modified to effectively activate B cells and upregulate their co-stimulatory molecules.

[0027] CD19 +"Lymphocytes": CD19 is a transmembrane protein specifically expressed on B lymphocytes and is a characteristic surface marker of B cells. Lymphocytes expressing CD19 are B lymphocytes, and can be called CD19⁺ B lymphocytes or CD19⁺ cells. CD19 is an inducible protein, and its elevated expression level can be used as a marker of B cell activation [Sato S, et al. Quantitative genetic variation in CD19 expression correlates with autoimmunity. J Immunol. 2000 Dec 1;165(11):6635-43]. CD19⁺ B lymphocytes express a variety of co-stimulatory molecules, including CD86, and elevated expression levels of these molecules also indicate B cell activation. CpG ODN can stimulate B cells to express co-stimulatory molecules. The antitumor deoxyribonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided in this invention can activate B lymphocytes and upregulate the expression of their co-stimulatory molecules.

[0028] "Co-stimulatory molecules": Co-stimulatory molecules are membrane proteins that provide an essential second signal for T cell activation, such as B7 family molecules (e.g., CD80, CD86) expressed on the surface of antigen-presenting cells. Effective T cell activation requires the simultaneous reception of both antigen recognition signals (the first signal) and co-stimulatory signals from T cell receptors; if antigen recognition is present but co-stimulation is lacking, not only will T cells not be adequately activated, but T cell dysfunction or immune tolerance may also be induced [Immunotherapy. 2014;6:1265–1278; Nat Rev Immunol. 2013;13:227–242]. In antitumor immunity, co-stimulatory signals are crucial for driving an effective T cell response.

[0029] “Co-inhibitory molecules”: Co-inhibitory molecules are a class of membrane proteins expressed on the surface of immune cells (such as T cells), such as PD-1 and CTLA-4. After binding to their corresponding ligands, they can transmit inhibitory signals, negatively regulating the intensity and duration of the immune response, and playing an important role in maintaining autoimmune tolerance and preventing damage to the body caused by excessive immune response. In the tumor microenvironment, tumor cells often use such molecular pathways (such as PD-L1 / PD-1) to inhibit T cell function, thereby achieving immune escape [Front Pharmacol. 2025 Jun 13;16:1602529].

[0030] Programmed cell death protein 1 (PD-1) is a transmembrane protein that is mainly expressed on the surface of activated CD8⁺T cells and CD4⁺T cells [Agata et al. IntImmunol. 1996;8:765-772; Yamazaki et al. J Immunol. 2002;169:5538-5545; Wherry et al. Immunity. 2007;27:670-684; Chikuma et al. J Immunol. 2009;182:6682-6689; Terawaki et al. J Immunol. 2011;186:2772-2779; Ahn et al. ProcNatl Acad Sci USA. 2018;115:4749-4754]. In addition, B cells, natural killer cells, NKT cells, dendritic cells and monocytes can also express PD-1 [Sharpe et al. Nat Immunol. 2007;8:239-245; Keir et al. Annu Rev Immunol. 2008;26:677-704]. PD-1 is a co-inhibitory molecule that participates in the establishment and maintenance of central and peripheral immune tolerance by regulating the intensity and quality of T cell responses [Nishimura et al. Immunity. 1999;11:141-151; Nishimura et al. Science. 2001;291:319-322; Wang et al. ProcNatl Acad Sci USA. 2005;102:11823-11828; Okazaki et al. Trends Immunol. 2006;27:195-201; Francisco et al. Immunol Rev. 2010;236:219-242; Fife et al. Ann NY Acad Sci. 2011;1217:45-59]. Resting dendritic cells can act on CD8⁺T cells through the PD-1 signaling pathway, thereby inducing immune tolerance in peripheral CD8⁺T cells [Probst et al. Nat Immunol. 2005;6:280-286].PD-1 modulates the intensity and duration of T cell responses [Okazaki et al. Nat Immunol. 2013;14:1212-1218; Honda et al. Immunity. 2014;40:235-247]. PD-1 functions as a co-inhibitory molecule in immune responses against pathogens and tumor cells [Attanasio et al. Immunity. 2016;44:1052-1068; Hashimoto et al. Annu Rev Med. 2018;69:301-318; Sharpe et al. NatRev Immunol. 2018;18:153-167]. PD-1 is an immune checkpoint protein, and its ligand is programmed death-ligand 1 (PD-L1).

[0031] Programmed death-ligand 1 (PD-L1): PD-L1 is a membrane protein [Blood. 2008 Apr 1;111(7):3635-43], expressed on the surface of most human and rodent cancer cells. PD-L1 can interact with PD-1, initiating inhibitory signaling pathways in T cells and interfering with activation signals for T cell activation, thereby suppressing T cell function [Honda et al., Immunity. 2014 Feb 20;40(2):235-247]. In the tumor microenvironment, cancer cells achieve immune evasion by overexpressing PD-L1 to suppress T cells. Anti-PD-L1 antibodies can block the binding of PD-L1 to PD-1 and reduce the inhibitory signals of T cell activation, thus restoring and enhancing the anti-tumor activity of T cells [Cell Rep. 2017 Aug 22;20(8):1818-1829; Nat Rev Dis Primers. 2020 May7;6(1):38]. Currently, the three humanized antibodies targeting PD-L1 that have been approved by the U.S. Food and Drug Administration are atezolizumab [Lancet. 2017 Jan 21;389(10066):255-265], durvalumab [N Engl J Med. 2017 Nov 16;377(20):1919-1929], and avelumab [Lancet Oncol. 2016 Oct;17(10):1374-1385]. They are used to treat a variety of malignant tumors, including non-small cell lung cancer, urothelial carcinoma, and triple-negative breast cancer.

[0032] "Immune checkpoints": Immune checkpoints (ICs) are a class of protein molecules that can transmit inhibitory signals to T lymphocytes after recognizing their corresponding ligands. When CD8⁺ T cells and CD4⁺ T cells are activated by dual signals, they express a variety of co-inhibitory molecules, including CTLA-4 and PD-1, on their surface. After these molecules bind to their ligands, they can transmit inhibitory signals into T cells, thereby limiting the further activation and function of T cells. These co-inhibitory molecules are collectively referred to as immune checkpoint proteins (IC proteins) [Ramos-Casals M, et al. NatRev Dis Primers. 2020 May 7;6(1):38]. In addition to CTLA-4 and PD-1, immune checkpoint proteins also include PD-L1 / PD-L2, lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), and B and T lymphocyte attenuator (BTLA). Immune checkpoint blockade refers to the inhibition of the transmission of inhibitory signals from immune checkpoint molecules to T cells, thereby removing their restriction on T cell activation. This strategy can enhance the body's immune response to tumor cells, thereby exerting an anti-tumor effect [Melero I, et al. Nat Rev Cancer. 2015 Aug;15(8):457-72].

[0033] "Immune checkpoint inhibitors": Immune checkpoint inhibitors (ICIs) are a class of formulations that block the transmission of inhibitory signals from immune checkpoint proteins to T cells. In this application, "formulation" and "drug" are used interchangeably and have the same or similar meanings. ICIs inhibit the inhibitory signals of anti-tumor T cells, keeping them in a fully activated state for a prolonged period to kill tumor cells, thus exhibiting anti-tumor efficacy. PD-L1 regulates T cell activity by transmitting inhibitory signals. Blocking this inhibitory signal may enable CD8⁺ T cells and CD4⁺ T cells to sustainably and efficiently kill tumor cells. PD-L1 antibodies can block this signaling pathway, thereby restoring and enhancing the sustained killing ability of CD8⁺ T cells and CD4⁺ T cells against tumor cells, exhibiting anti-tumor activity. PD-L1-specific antibodies that can block PD-L1 transmission have become an anti-tumor drug. This drug is an immune checkpoint inhibitor (ICI) [Schaub J, Tang SC. Beyond checkpoint inhibitors: the three generations of immunotherapy. Clin Exp Med. 2025 Jan 21;25(1):43]. ICIs work by "inhibiting inhibition to maintain activation." PD-L1 antibodies recognize and bind to PD-L1, blocking the transduction of inhibitory signals that suppress T lymphocyte activation, thereby keeping T cells in a state of sustained and adequate activation. The antitumor deoxyribonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided in this invention is a novel ICI.

[0034] "Immune checkpoint antibodies": Immune checkpoint antibodies (ICI antibodies) are a class of antibodies that can target and bind to immune checkpoint proteins, thereby blocking the transmission of inhibitory signals to T lymphocytes. Humanized monoclonal antibodies targeting CTLA-4, PD-1, and PD-L1 belong to this class. Currently, the US FDA has approved the following eight immune checkpoint antibodies for cancer treatment: Ipilimumab, targeting CTLA-4, is indicated for melanoma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer, and malignant pleural mesothelioma; Pembrolizumab, targeting PD-1, is indicated for melanoma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, urothelial carcinoma, gastric cancer, esophageal cancer, Merkel cell carcinoma, endometrial cancer, MSI-H cancer, squamous cell carcinoma of the skin, cervical cancer, head and neck squamous cell carcinoma, and triple-negative breast cancer; Nivolumab, targeting PD-1, is indicated for melanoma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, and non-small cell lung cancer. Cellular lung cancer, small cell lung cancer, classical Hodgkin lymphoma, urothelial carcinoma, esophageal cancer; Atezolizumab, targeting PD-L1, is indicated for melanoma, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, urothelial carcinoma; Avelumab, targeting PD-L1, is indicated for Merkel cell carcinoma, urothelial carcinoma, renal cell carcinoma; Durvalumab, targeting PD-L1, is indicated for non-small cell lung cancer, urothelial carcinoma, small cell lung cancer; Cemiplimab, targeting PD-1, is indicated for squamous cell carcinoma of the skin, basal cell carcinoma, non-small cell lung cancer; Dostarlimab, targeting PD-1, is indicated for recurrent or advanced solid tumors with mismatch repair deficiency (dMMR) [Schaub J, Tang SC. ClinExp Med. 2025;25(1):43]. The anti-tumor deoxy oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided by this invention can be used in combination with immune checkpoint antibodies to treat tumors.

[0035] "Immune-related adverse events": The use of immune checkpoint inhibitors (such as CTLA-4 antibodies, PD-1 antibodies, and PD-L1 antibodies) to treat tumors can trigger a variety of diseases, which are defined as immune-related adverse events (irAEs). irAEs can affect almost all organ systems [Puzanov I, et al. JImmunother Cancer. 2017;5(1):95; Ramos-Casals M, et al. Nat Rev Dis Primers.2020;6(1):38]. Most irAEs are mild to moderate, but there are also life-threatening severe cases, such as severe colitis, pneumonia, encephalitis, toxic epidermal necrolysis, myocarditis, and type 1 diabetic ketoacidosis [Puzanov I, et al. JImmunother Cancer. 2017;5(1):95]. Nivolumab is a human IgG4 antibody targeting PD-1 [Seidel et al. Front Oncol. 2018;8:86], which has been approved for the treatment of metastatic melanoma, metastatic non-small cell lung cancer (NSCLC), and hepatocellular carcinoma (HCC) [Sundar et al. Ther Adv Med Oncol.2015;7(2):85–96; Guntur et al. Expert Opin Investig Drugs. 2015;24(2):253–26; Finkelmeier et al. Expert Rev Anticancer Ther. 2018;18(12):1169–1175]. Multiple irAEs, including pneumonia, encephalitis and nephritis, have been observed in patients receiving nivolumab treatment [Wang et al. Technol Cancer Res Treat. 2020;19:1533033820967454; Palmieri et al. CurrOncol Rep. 2018;20(9):72; Sundar et al. Ther Adv Med Oncol. 2015;7(2):85–96].Pembrolizumab is a humanized IgG4 antibody that also targets PD-1 and is indicated for the treatment of non-small cell lung cancer (NSCLC), classical Hodgkin lymphoma [Seidel et al. Front Oncol. 2018;8:86], metastatic anal cancer, refractory esophageal cancer, and metastatic HER2-negative breast cancer [Rugo et al. Clin Cancer Res.2018;24(12):2804–2811;Doi T et al. J Clin Oncol. 2018;36(1):61–67;Marabelle et al. J Clin Oncol. 2020;38(4):1.31682550;Phuong et al. Curr Oncol Rep.2020;22(9):9]. Pembrolizumab can cause a range of adverse reactions, including fatigue, endocrine disorders, hepatitis, and pneumonia [Wang et al. Technol Cancer Res Treat. 2020;19:1533033820967454; Palmieri et al. Curr Oncol Rep. 2018;20(9):72]. The overall incidence of cardiotoxicity after using PD-1 antibodies is less than 1%, but it can be flare-ups, progressive, and fatal. Combination therapy with immune checkpoint antibodies is more likely to cause myocarditis. This myocarditis usually appears within about 4 weeks after the first dose [Ramos-Casals M, et al. Nat Rev Dis Primers. 2020 May 7;6(1):38]. PD-1 antibodies can also cause skin lesions, with symptoms usually appearing about 5 weeks after the start of treatment [Ramos-Casals M, et al. Nat Rev Dis Primers. 2020 May 7;6(1):38]. In patients receiving anti-PD-1 antibodies, the incidence of endocrine-related irAEs (such as hypophysitis and thyroid dysfunction) is 4%–14% [Ramos-Casals M, et al. Nat Rev DisPrimers. 2020 May 7;6(1):38], and the incidence of pneumonia is approximately 1%–3%. The antitumor deoxygenated oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided in this invention is a non-antibody ICI targeting PD-L1, which largely avoids the occurrence of antibody-dependent irAEs.

[0037] “Anti-tumor single-stranded deoxyoligonucleotides”: “Anti-tumor single-stranded deoxyoligonucleotides” refers to a class of single-stranded deoxyoligonucleotides with specific base sequences that can activate the body’s innate immune response and cellular immune response against tumor cells, thereby potentially exerting an anti-tumor effect [Akira S, et al. Nature. 2000;408:740–745; Klinman DM. Nat Rev Immunol. 2004;4:249–259; KriegAM, et al. J Clin Invest. 2007;117:1184–94; Scheiermann J, et al. Vaccine.2014 Nov 12;32(48):6377–89].Based on the aforementioned immunomodulatory activities, these oligonucleotides are expected to be used as adjuvants for tumor vaccines, and can also be used alone or in combination with other antitumor drugs for tumor treatment [Shi S, et al. Vaccine. 2019 May 27;37(24):3167-3178;Karbach J, et al. Int J Cancer. 2010;126:909–18;Speiser DE, et al. J Clin Invest. 2005;115:739–46;Valmori D, et al. Proc Natl Acad Sci US A. 2007;104:8947–52;Carpentier A, et al. NeuroOncol. 2010;12:401–8;Hirsh V, et al. J Clin Oncol. 2011;29:2667–2674;SmithDA, et al. Cancer Immunol Immunother. 2014;63:787–796; Chan E, et al. CancerChemother Pharmacol. 2015;75:701–9; Friedberg JW, et al. Br J Haematol. 2009;146:282–91; Brody JD, et al. Blood. 2009;113:85–94; Lim SH, et al. ImmunolRef. 2014;193:1519–24; Marabelle A, et al. 2014;20:1747–56; Zent CS, et al. Leuk Lymphoma. 2012;53:211–7; Link BK, et al. Cancer. 2019 Nov 26;7(1):323;Mangsbo SM, et al. IntImmunol. 2010;22:651–60].

[0038] "Anti-tumor drug": In this invention, "anti-tumor drug" and "anti-tumor agent" are used interchangeably and have the same or similar meanings. The anti-tumor drugs include, but are not limited to, the following categories: chemotherapy drugs, tumor vaccines, therapeutic antibodies, immune checkpoint antibodies, tumor angiogenesis inhibitors, oncolytic viruses, small molecule kinase inhibitors, and cells used for cell therapy (such as dendritic cells, CAR-T cells, tumor-infiltrating lymphocytes, and natural killer cells). The anti-tumor deoxyribonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided by this invention can be used in combination with these anti-tumor drugs to enhance their tumor therapeutic effects.

[0039] "Tumor vaccine": A tumor vaccine is an immunomodulatory agent prepared by combining tumor antigens and vaccine adjuvants [DeMaria PJ, et al. Hematol Oncol Clin North Am. 2019 Apr;33(2):199-214]. Its mechanism of action is to activate the body to produce an anti-tumor immune response, thereby inhibiting or eliminating tumor cells. The anti-tumor deoxygenated oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided in this invention can be used in combination with tumor vaccines to enhance their efficacy in inducing anti-tumor immunity. Types of tumor vaccines include tumor antigen peptide vaccines, DNA or mRNA vaccines encoding tumor antigens, dendritic cell vaccines loaded with tumor antigens, and viral vector vaccines carrying tumor antigen encoding genes [Saxena M, et al. Nat Rev Cancer. 2021 Jun;21(6):360-378]. The mechanism of action of tumor vaccines mainly includes the following steps: (1) Antigen presentation: The tumor antigens in the vaccine are taken up and processed by antigen-presenting cells (such as dendritic cells), and then presented to their surface through MHC molecules. (2) T cell activation: The presented antigens are recognized by T cells, thereby activating tumor-specific T cells. B lymphocytes also promote the activation of anti-tumor T cells. Dendritic cells and B cells activated by CpG ODN are more effective in activating anti-tumor T cells. (3) Tumor killing: The activated T cells migrate to the site where tumor cells are present, recognize and kill tumor cells expressing the corresponding antigens.

[0040] "Tumor antigens": Tumor antigens can stimulate an individual to produce a tumor-specific immune response, thereby exerting an anti-tumor therapeutic effect [Finn OJ. Cancer Immunol Res. 2017 May;5(5):347-354]. Tumor antigens include tumor-associated antigens (TAAs), tumor-specific antigens, oncogenic viral antigens, and neoantigens [Hu Z, et al. Nat Rev Immunol. 2018 Mar;18(3):168-182; Sahin U, et al. Science. 2018 Mar 23;359(6382):1355-1360; Ott PA, et al. Nature. 2017 Jul 13;547(7662):217-221]. The antitumor deoxy oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided by this invention can enhance the body's adaptive immune response to tumor antigens.

[0041] Neoantigens, also known as tumor neoantigens, are a class of antigens produced by specific mutations in tumor cells. These antigens typically originate from genetic variations in tumor cells, such as gene mutations, insertions / deletions, gene rearrangements, or aberrant splicing. These variations alter protein sequences, resulting in new peptides. These new peptides can be presented to the cell surface by major histocompatibility complex (MHC) molecules for recognition by T cells. Unlike tumor-associated antigens, neoantigens are expressed only in tumor cells. Therefore, immune responses against neoantigens can more precisely target tumor cells and reduce damage to normal tissues. Based on these characteristics, neoantigens can be used to develop personalized tumor vaccines. These vaccines are customized based on the patient's tumor-specific antigens and can activate a specific immune response against the tumor. Their design depends on the patient's gene mutation profile or the antigen information of the tumor [Hu Z, et al. Nat Rev Immunol. 2018 Mar;18(3):168-182 Sahin U, et al. Science. 2018 Mar 23;359(6382):1355-1360;Ott PA, et al. Nature. 2017 Jul 13;547(7662):217-221].

[0042] "Tumor vaccine adjuvants": Tumor vaccine adjuvants are substances that enhance the immunogenicity of tumor antigens when used in conjunction with them. Tumor vaccine adjuvants include various Toll-like receptor agonists, such as: polyinosinic-cytosine nucleotide (polyIC) and its derivative poly-ICLC (polyinosinic–polycytidylic acid with polylysine and carboxymethylcellulose) as TLR3 agonists; monophosphoryl lipid A (MPL) as a TLR4 agonist; imiquimod as a TLR7 agonist; resiquimod acting on TLR7 / 8; and CpG ODN (CpG oligodeoxynucleotide) as a TLR9 agonist. In addition, tumor vaccine adjuvants also include cyclic dinucleotides and their analogues (such as cyclic di-guanosine monophosphate) and BCG [Maisonneuve C, et al. Proc Natl Acad Sci US A.2014 Aug 26;111(34):12294-9; Campbell JD. Methods Mol Biol. 2017;1494:15-27;Ablasser A, Chen ZJ. Science. 2019 Mar 8;363(6431):eaat8657;Chandra D et al. Cancer Immunol. Res 2, 901–910 (2014)]. The antitumor deoxy oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided in this invention can be used as a tumor vaccine adjuvant and can be used in combination with other types of adjuvants to treat tumors.

[0043] "tumor therapeutic antibodies": Tumor therapeutic antibodies are antibodies that can have a therapeutic effect on individual tumors [Scott AM et al. Nat Rev Cancer. 2012 Mar 22;12(4):278-87]. This class of antibodies targets different sites, such as: (1) Tositumomab (Bexxar), Rituximab (Rituxan) and Ofatumumab (Arzerra; Genmab) targeting CD20; (2) Trastuzumab (Herceptin) targeting ErbB2; (3) Panitumumab (Vectibix) and Cetuximab (Erbitux) targeting epidermal growth factor receptor; (4) Bevacizumab targeting vascular endothelial growth factor; (5) Brentuximab vedotin targeting CD30; (6) Alemtuzumab (Campath) targeting CD52 and Gemtuzumab ozogamicin targeting CD33; (7) Antibodies targeting immune checkpoints [Schaub J, Tang SC. Clin Exp Med. 2025 Jan 21;25(1):43]. The anti-tumor deoxy oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided by this invention can be used in combination with the above-mentioned tumor therapeutic antibodies to treat tumors.

[0044] "Anti-tumor angiogenesis drugs": These drugs inhibit tumor angiogenesis by blocking the blood supply to the tumor, thereby suppressing the growth and metastasis of tumor cells. These drugs primarily target vascular endothelial growth factor (VEGF) and proteins involved in its signaling pathway. Bevacizumab (Avastin®) is a monoclonal antibody targeting VEGF-A and was the first approved anti-tumor angiogenesis drug. It was initially approved in combination with chemotherapy for the treatment of metastatic colorectal cancer, and its indications have since expanded to include metastatic breast cancer, non-small cell lung cancer, glioblastoma, renal cell carcinoma, ovarian cancer, and cervical cancer [Garcia J, et al. Cancer Treat Rev. 2020 Jun;86:102017]. Many small molecule tyrosine kinase inhibitors, such as sunitinib and sorafenib, inhibit multiple targets, including the VEGF receptor, and also belong to the category of anti-tumor angiogenesis drugs. The antitumor deoxy oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided by this invention can be used in combination with drugs that inhibit tumor angiogenesis to treat tumors.

[0045] "Bispecific antibodies for tumor therapy": Bispecific antibodies for tumor therapy are genetically engineered recombinant immunoglobulins. Their structural feature is that they contain two different antigen-binding domains, enabling them to simultaneously and specifically recognize and bind to two different antigenic epitopes. Bispecific antibodies for tumor therapy are bispecific antibodies (bsAbs). As of the end of 2023, 11 bispecific antibodies have been approved for cancer treatment [Klein C, et al. The present and future of bispecific antibodies for cancer therapy. Nat Rev DrugDiscov. 2024 Apr;23(4):301-319]. The antitumor deoxygenated oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided by this invention can be used in combination with bispecific antibodies for tumor therapy to treat tumors.

[0046] Oncolytic viruses are genetically engineered or naturally occurring viral strains that can selectively replicate and lyse tumor cells without affecting normal cells. Their mechanisms of action include: recognizing and invading tumor cells through specific viral receptors; selectively replicating using abnormal signaling pathways within tumor cells; and ultimately directly destroying tumor cells through cell lysis. Viral proteins expressed during replication can also activate the body's anti-tumor immune response, including the release of tumor-associated antigens and danger signaling molecules, thereby promoting antigen presentation and T-cell activation. Representative oncolytic viruses include genetically engineered strains of herpes simplex virus, adenovirus, and vaccinia virus [Kaufman HL, et al. Nat Rev Drug Discov. 2015 Sep;14(9):642-62; June CH, et al. N Engl J Med. 2018 Jul 5;379(1):64-73]. Talimogene laherparepvec is the first approved type 1 herpes simplex oncolytic virus for the treatment of recurrent melanoma, and its genome carries the coding sequence for human granulocyte-macrophage colony-stimulating factor [Wang H, et al. J Immunother Cancer. 2024 May 31;12(5):e008025]. The antitumor deoxygenated oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided in this invention can be used in combination with oncolytic viruses to treat tumors.

[0047] Small molecule kinase inhibitors: Small molecule kinase inhibitors are chemically synthesized low-molecular-weight compounds whose mechanism of action is to specifically target and inhibit the activity of protein kinases that play a key role in tumor cell signal transduction. These kinases include, but are not limited to, receptor tyrosine kinases (such as EGFR, VEGFR, PDGFR) and non-receptor tyrosine kinases (such as BCR-ABL), as well as serine / threonine kinases (such as mTOR, AKT). These inhibitors block the kinase-mediated downstream signaling pathways by competing for ATP binding sites or through allosteric regulation, thereby inhibiting the abnormal proliferation of tumor cells, promoting apoptosis, or inhibiting tumor angiogenesis. Tyrosine kinase inhibitors (TKIs) are a class of small molecule kinase inhibitors. TKIs targeting Bcr-ABL, epidermal growth factor receptor, and vascular endothelial growth factor receptor have been used to treat various tumors [Hussain S, et al. Eur J Pharmacol. 2024 May 5;970:176484]. The antitumor deoxy oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided by this invention can be used in combination with small molecule kinase inhibitors to treat tumors.

[0048] “Cells for Tumor Therapy”: Cells for tumor therapy include dendritic cells [Nestle, F. et al. (1998) Nature Medicine 4: 328-332; Palucka K et al. Nat Rev Cancer. 2012 Mar 22;12(4):265-77; Kugler, A. et al. (2000) Nature Medicine 6:332-336; Stevens D, et al. Front Immunol. 2021 Feb 12;11:620374; Gardner A, et al. Front Immunol.2020 May 21;11:924] and genetically engineered T cells expressing chimeric antigen receptors (CARs) (CAR-T cells) [Kershaw MH et al. Nat Rev Cancer. 2013 Aug;13(8):525-41; Hong M, et al. Cancer Cell. 2020] Oct 12;38(4):473-488], tumor-infiltrating lymphocytes [Rosenberg SA, et al. N Engl J Med. 1988 Dec 22;319(25):1676-80], and natural killer cells [Childs RW, et al. Nat Rev Drug Discov. 2015 Jul;14(7):487-98]. Currently, the U.S. Food and Drug Administration (FDA) has approved six CAR-T cell therapy products for the treatment of B-cell acute lymphoblastic leukemia, large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, and multiple myeloma [Brudno JN, et al. JAMA. 2024 Dec 10;332(22):1924-1935]. The anti-tumor deoxy oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided by this invention can be used in combination with tumor therapeutic cells to treat tumors.

[0049] "Synthetic lethality in cancer cells": Synthetic lethality refers to the genetic phenomenon where the simultaneous loss of function of two genes leads to cell death, while the loss of either gene alone allows the cell to survive. Based on this phenomenon, therapeutic strategies to induce synthetic lethality in cancer cells have been designed, which involves using two or more methods or drugs to kill cancer cells. In preclinical experiments, the combined use of PLX4032 (a BRAF inhibitor, encofenib) and cetuximab (an EGFR-targeting antibody, cetuximab) has shown that it can effectively kill tumor cells. PLX4032 (a BRAF inhibitor) and gefitinib (an EGFR inhibitor) have also shown that they can effectively kill tumor cells [Prahallad, A. et al. Nature. 483, 100–103 (2012)]. In 2020, the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) approved the combination of the BRAF inhibitor encorafenib and the EGFR-targeting antibody cetuximab for the treatment of BRAF-mutant metastatic colorectal cancer as a synthetic lethal therapy. Synthetic lethal strategies for cancer cells include dual synthetic lethality, multi-synthetic lethality, and compound synthetic lethality [Ryan CJ, et al. Nat Genet. 2023 Dec;55(12):2039-2048]. In October 2022, the U.S. FDA approved the combination of tremelimumab (anti-CTLA-4 antibody) and durvalumab (anti-PD-L1 antibody) for the treatment of unresectable hepatocellular carcinoma. This is also a dual synthetic lethal therapy for cancer cells [Sho T, et al. Target Oncol. 2024 Sep;19(5):769-778]. The anti-tumor deoxy oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided by this invention can be used in combination with anti-tumor agents (drugs) to treat tumors and promote the synthetic lethality of cancer cells.

[0050] "Local tumor administration": Local tumor administration refers to the application of antitumor drugs via intratumoral injection, peritumoral injection, injection into the tumor drainage lymph node area, interventional embolization, implantation of sustained-release agents, or local perfusion. Local tumor administration may improve the efficacy of antitumor drugs and reduce side effects [Fransen MF, et al. Clin. Cancer Res 19, 5381–5389(2013); Marabelle A, et al. Clin. Cancer Res 19, 5261–5263 (2013)]. Direct injection of micrograms of CpG ODN into mouse tumor lesions helps to form an antitumor immune microenvironment and trigger a systemic antitumor immune response [Sagiv-Barfi I, et al. Sci Immunol. 2022 May 27;7(71):eabn5859]. The antitumor deoxygenated oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided in this invention can be applied via the local tumor administration route.

[0051] "Route of Administration for Antitumor Drugs": This invention relates to routes of administration for antitumor drugs, including but not limited to systemic and local administration. Systemic administration routes include intravenous injection, arterial injection, intramuscular injection, subcutaneous injection, and oral administration. Local administration routes include intracavitary perfusion (such as intraperitoneal or intrapleural perfusion), intrathecal injection, and local tumor administration. The antitumor deoxyribonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided by this invention can be applied via the routes of administration for antitumor drugs.

[0052] "Antitumor Drug Delivery System": The antitumor deoxy oligonucleotide (ILO) containing CpG motifs that interferes with PD-L1 expression provided by this invention can be delivered using an antitumor drug delivery system. The antitumor drug delivery system includes, but is not limited to, lipid nanoparticles (LNPs), cationic lipid complexes, emulsifiers, liposomes, polymer nanoparticles, targeted nanocarriers, scaffolds, and hydrogels [Cullis PR, Felgner PL. Nat RevDrug Discov. 2024 Sep;23(9):709-722; Ramos-Casals M, et al. Nat Rev DisPrimers. 2020 May 7;6(1):38]. Attached Figure Description

[0053] Figure 1 Figure 1 shows the activation effect of antitumor deoxy oligonucleotides (ILOs) containing CpG motifs that interfere with PD-L1 expression on B cells.

[0054] Figure 2 Figure 1 shows the inhibitory effect of antitumor deoxygenated oligonucleotides (ILOs) containing CpG motifs on PD-L1 expression in tumor cells.

[0055] Figure 3 Figure 1 shows the inhibitory effect of antitumor deoxygenated oligonucleotides (ILOs) containing CpG motifs on PD-L1 expression in immune cells.

[0056] Figure 4 Figure 1 shows the inhibitory effect of CpG motif-containing interfering PD-L1 expression-disrupting deoxyribonucleotides (ILOs) on the growth of melanoma formed from B16 cells.

[0057] Figure 5 The figure shows the inhibitory effect of CpG motif-containing interfering PD-L1 expression-disrupting deoxyribonucleotides (ILOs) on tumor growth in lung cancer cells by activating TLR9 and inhibiting PD-L1.

[0058] Figure 6 The effect of antitumor deoxyribonucleotides (ILOs) containing CpG motifs that interfere with PD-L1 expression on tumor tissue cells and draining lymph node cells in Lewis lung cancer cells-forming mice is shown in the figure. Specific implementation methods:

[0059] Example 1: Design, synthesis, dissolution, and endotoxin content detection of an antitumor deoxy oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression.

[0060] Having a sequence list <400> 1 (SEQ ID NO: 1) shows a single-stranded deoxynucleotide (ODN) with the sequence (5'-CTTGTCACGCTCAGCCTCGATGAA-3'), which is named ILO. ILO is a single-stranded deoxynucleotide with the sequence (5'-CTTGTCACGCTCAGCCTCGATGAA-3'). No-CpG-ILO (5'-CTTGTCAGCCTCAGCCCCCATGAA-3') is a control oligonucleotide that retains the common ILO sequence but lacks the CpG motif. No-ASO-ILO (5'-GAACAGTGCGAGTCGGAGCTACTT-3') is an ILO sense control oligonucleotide that retains the CpG motif. CpG 5805 (5'-TCGACGAACGTTCGGTCGCCGCGG-3') is a C-type CpG oligonucleotide. These oligonucleotides were synthesized and purified by Takara Bio Engineering (Dalian) Co., Ltd. All oligonucleotides were fully phosphorothioated for internucleotide linkages. The oligonucleotides were diluted with PBS (biopico, catalog number #090501) and tested negative for endotoxins using a horseshoe crab lysate assay (Associates of Cape Cod, Inc., catalog number #PSD250-10). They were aliquoted and stored at -20 °C for later use.

[0061] Example 2: Activation effect of CpG motif-containing antitumor deoxy oligonucleotides (ILO) that interfere with PD-L1 expression on B cells.

[0062] 2.1. Materials

[0063] Female BALB / c mice aged 6-8 weeks (RRID: IMSR_CRL:028) were purchased from Yisi Laboratory Animal Technology Co., Ltd., Changchun, China. These mice were housed under specific pathogen-free (SPF) conditions at the Experimental Animal Center of Jilin University, with free access to food and water. All animal experiments were conducted in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals (GB / T 35892-2018) and were approved by the Ethics Committee of the School of Basic Medical Sciences, Jilin University (Approval No.: 2024614). All procedures were performed in accordance with relevant guidelines and regulations.

[0064] Single-stranded deoxy oligonucleotides (ILOs) having the sequence (5'-CTTGTCACGCTCAGCCTCGATGAA-3') as described in Example 1.

[0065] CpG 5805 (5'-TCGACGAACGTTCGGTCGCCGCGG-3').

[0066] The culture media used for cell culture were as follows: Complete RPMI medium: based on RPMI 1640 (Gibco, cat#C11875500BT), supplemented with 10% (v / v) fetal bovine serum (FBS) (Gibco, cat#C11875500BT), 100 IU / mL penicillin and 100 IU / mL streptomycin (Gibco, cat#15140148); Serum-free RPMI medium: without FBS, containing 100 IU / mL penicillin and 100 IU / mL streptomycin; RPMI medium with 2% FBS: containing 2% (v / v) FBS, 100 IU / mL penicillin and 100 IU / mL streptomycin.

[0067] Antibodies used for flow cytometry analysis: CD19-PE (BD, Cat# 557399), CD86-APC (BD, Cat# 558703).

[0068] 2.2. Methods

[0069] like Figure 1 As shown in Figure A, 6-8 week old BALB / c mice (n=3) were euthanized and immersed in 75% ethanol for 2 minutes. Subsequently, the spleens were removed using ophthalmic scissors and forceps. A single-cell suspension was prepared by grinding the cells on ice using a ground glass slide in pre-chilled complete RPMI-1640 medium (with 10% FBS). The suspension was filtered through a 300-mesh sieve and centrifuged at 900 rpm for 5 minutes. The cell pellet was treated on ice for 2 minutes with 3 mL of potassium ammonium chloride (ACK) lysis buffer (NH4Cl 8.024 mg / L, KHCO3 1001 mg / L, Na2EDTA 3.7 mg / L, pH 7.2–7.4) (pH 7.2–7.4). Lysis was terminated by adding 5 mL of PBS, followed by centrifugation. The cells were then resuspended in complete medium for counting. The counted spleen cells (3 × 10⁶) were... 6Cells were seeded at 1 mL per well in 24-well plates (NEST, China) with ILO (3 μg / mL), CpG 5805 (3 μg / mL), or RPMI 1640 medium (Med), and co-cultured at 37°C in a 5% CO2 incubator for 24 hours. Cells were collected, centrifuged, and resuspended in 50 μL of PBS containing 2% FBS for antibody staining. The CD19-PE (BD, catalog number 557399) and CD86-APC (BD, catalog number 558703) antibodies used were diluted 1:10. Isotype controls were set up to assess non-specific binding. Stained cells were analyzed on a BD Accuri C6 Plus flow cytometer, and raw data were processed using BD Accuri C6 Plus software version 1.0.23.1. Viable cells were gated using the P1 gate (P1). From P1, cells expressing CD19 (CD19⁺ cells) (R1) and cells highly expressing CD19 (CD19⁺ cells) were further sorted. hi Cells) (R2) Figure 1 A). ( Figure 1 B) Shows CD19⁺ cells or CD19 hi Percentage of cells in total spleen cells (P1) (%). Figure 1 C) The figure shows the percentage (%) of CD86 and CD19 co-expressing cells (CD86⁺CD19⁺ cells) in CD19⁺ cells (R1), or the percentage of CD19⁺ cells co-expressing cells (CD86⁺CD19⁺ cells). h CD86 and CD19 in i cells (R2) hi Co-expressing cells (CD86-CD19) hi Percentage of cells (%). Data are expressed as mean ± standard deviation. Unpaired t-tests were used to determine significance; *, **, and *** represent p < 0.05, 0.01, and 0.001, respectively.

[0070] 2.3 Results

[0071] like Figure 1 As shown, mouse spleen cells stimulated with an anti-tumor deoxyribonucleic acid (ILO) containing a CpG motif that interferes with PD-L1 expression showed a high expression of CD19 (CD19). hi The number of cells increased significantly. Figure 1 B); The number of CD86 and CD19 co-expressing cells (CD86⁺CD19⁺ cells) in mouse spleen cells significantly increased after ILO stimulation. Figure 1 C); CD86 and CD19 in mouse spleen cells after ILO stimulation hi Co-expressing cells (CD86-CD19) hi The number of cells increased significantly.

[0072] These results ( Figure 1 This indicates that: (1) ILO can induce B cells to express CD19; (2) ILO can induce the production of B cells that highly express CD19 (i.e., CD19). hi (3) ILO can induce B cells to express CD86; (4) ILO can promote B cell proliferation. CD19 is a transmembrane protein expressed on B lymphocytes and is a specific surface marker of B cells. Lymphocytes that express CD19 on their surface are B lymphocytes, so B cells are often referred to as CD19 cells. + B lymphocytes or CD19 + B cells. CD19 is an inducible protein, and its upregulation is a typical phenotypic feature of B cell activation [Sato S, et al. J Immunol. 2000 Dec 1;165(11):6635-43; Szelinski F, et al. ArthritisRheumatol. 2022 Sep;74(9):1556-1568]. As a co-stimulatory molecule of the B cell receptor (BCR), CD19 can enhance BCR-mediated signal transduction. Studies have shown that the activation effect of TLR9 agonists on B cells depends on CD19 [Morbach H, et al. J Allergy Clin Immunol. 2016;137(3):889–98.e6]. Overexpression of CD19 further enhances BCR-mediated activation signals [Inaoki M, et al. J Exp Med. 1997 Dec 1;186(11):1923-31]. In mouse models, B cells highly expressing CD19 can more effectively activate CD8+ T cells and enhance their ability to kill target cells [Ziegler AI, et al. Diabetologia. 2013 Dec;56(12):2659-68]. The antitumor deoxyribonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided in this invention can induce B cells to express CD19 and increase the number of B cells with high levels of CD19 expression (CD19... hi The invention can increase the number of B cells and induce B cells to express CD86. High CD86 expression in B cells provides a stronger co-stimulatory signal to T cells, thereby enhancing T cell activation. These results indicate that the anti-tumor deoxygenated oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided in this invention can activate B cells, enabling them to more effectively synergize with anti-tumor T cells to kill tumor cells, thus exerting a therapeutic effect on tumors.

[0073] These results ( Figure 1 This also indicates that ILO is a TLR9 agonist. The core mechanism by which TLR9 agonists exert their anti-tumor effects lies in activating innate immunity and promoting adaptive anti-tumor immune responses. TLR9 is mainly expressed in immune cells such as plasmacytoid dendritic cells (pDCs) and B cells. When oligonucleotides containing CpG motifs are recognized by TLR9 as agonists, they activate the MyD88-dependent signaling pathway, inducing the production of large amounts of type I interferons (such as IFN-α) and pro-inflammatory cytokines (such as IL-12). This directly inhibits tumor cell proliferation and activates innate immune cells such as natural killer (NK) cells to exert early killing effects. At the same time, cytokines such as IL-12 and interferons can promote the maturation and function of antigen-presenting cells (such as dendritic cells), thereby effectively activating and guiding antigen-specific CD4⁺T cells and CD8⁺T cells to respond. Thus, the activation of innate immunity is transformed into a powerful and tumor antigen-targeting specific cellular immunity, ultimately achieving long-term immune surveillance and clearance of tumor cells.

[0074] Example 3: Inhibitory effect of CpG motif-containing antitumor deoxygenated oligonucleotides (ILO) on PD-L1 expression in B16 melanoma cells.

[0075] 3.1. Materials

[0076] B16 melanoma cells (B16 cells; RRID: CVCL_0158) (purchased from the American Type Culture Collection) were cultured in RPMI-1640 medium (Gibco, catalog number C11875500BT) supplemented with 10% heat-inactivated fetal bovine serum (FBS; OriCell, catalog number FBSST-01033-500) and antibiotics (penicillin and streptomycin, 100 IU / mL each; Gibco, catalog number 15140148). Cells were maintained in a humidified environment at 37°C and 5% CO2, and were used within 2 months after resuscitation, with a passage count not exceeding 20 times. The B16 cells were maintained by the Department of Immunology, Jilin University, and were regularly screened for mycoplasma contamination using a mycoplasma detection kit (Yeasen Biotech, catalog number 40612ES25), with consistently negative results.

[0077] Single-stranded deoxy oligonucleotides (ILOs) with the sequence (5'-CTTGTCACGCTCAGCCTCGATGAA-3') as described in Example 1. LPS (Yeasen, Cat#60748ES).

[0078] Antibody for flow cytometry analysis: CD274-PE (Invitrogen, Cat#2284281).

[0079] 3.2. Methods

[0080] like Figure 2 As shown, B16 melanoma cells (B16 cells) (2 × 10⁻⁶) 5 / ml) were co-cultured with the following conditions: RPMI 1640 medium (Med), Med plus ILO (3 μg / ml) (Med+ILO) ( Figure 2 A), or co-cultured with Med, Med plus LPS (Yeasen, Cat#60748ES) (5 μg / ml) (Med+LPS), Med plus LPS (5 μg / ml) plus ILO (3 μg / ml) (Med+LPS+ILO) ( Figure 2 B). After 24 hours of incubation, the cell suspension was collected into 1.5 ml Eppendorf tubes and centrifuged at 3000 rpm for 5 minutes, discarding the supernatant. Cells were resuspended in 50 μl of PBS (Biopico, China) containing 2% FBS, and PD-L1 antibody CD274-PE (Invitrogen, Cat#2284281) diluted 1:10 in PBS was added. The cells were incubated on ice in the dark for 30 minutes. After incubation, cells were washed with PBS, centrifuged at 3000 rpm for 5 minutes, and the supernatant was discarded. Finally, cells were resuspended in 200 μl of PBS containing 2% FBS. An isotype control was used to assess nonspecific binding. Finally, cell analysis was performed using flow cytometry (BD Biosciences, Accuri C6 Plus, RRID: SCR_019591), and the raw data were analyzed using BD Accuri C6 Plus software 1.0.23.1. Using the live cell population P1 as a gate, the expression level of PD-L1 in B16 cells (quantified by mean fluorescence intensity MFI) and the frequency of PD-L1⁺ cells in B16 cells were further analyzed. Data are expressed as mean ± standard deviation. Statistical significance was assessed using an unpaired t-test. *, **, and *** represent p < 0.05, 0.01, and 0.001, respectively.

[0081] 3.3 Results

[0082] like Figure 2 As shown, antitumor deoxyribonucleotides (ILOs) containing CpG motifs that interfere with PD-L1 expression significantly inhibited PD-1 expression in tumor cells and reduced the number of tumor cells expressing PD-1. Figure 2(B) This indicates that ILO exerts its anti-tumor effect by inhibiting PD-L1 expression on tumor cells. PD-L1 expression on the surface of tumor cells is a key mechanism for their immune evasion. By binding to PD-1 on the surface of T cells, it transmits inhibitory signals, leading to T cell inactivation or exhaustion, thereby escaping immune attack. ILO, through its antisense oligonucleotide function, precisely targets and downregulates PD-L1 expression in tumor cells, directly blocking this immunosuppressive pathway. Its core mechanism lies in relieving tumor-mediated immunosuppression: when PD-L1 expression is inhibited, tumor cells lose their effective suppression of tumor-infiltrating T cell activity. This is equivalent to removing the inhibitory signal from the immune system, allowing previously suppressed effector cells such as CD8⁺ T cells to regain their killing ability, thus more effectively recognizing and eliminating tumor cells. Therefore, ILO weakens the tumor's immune resistance at its source by directly targeting the immune checkpoint molecules of tumor cells.

[0083] Example 4: Inhibitory effect of CpG motif-containing antitumor deoxygenated oligonucleotides (ILO) on PD-L1 expression in T cells.

[0084] 4.1. Materials

[0085] Female BALB / c mice aged 6-8 weeks (RRID: IMSR_CRL:028) were purchased from Yisi Laboratory Animal Technology Co., Ltd., Changchun, China. These mice were housed under specific pathogen-free (SPF) conditions at the Experimental Animal Center of Jilin University, with free access to food and water. All animal experiments were conducted in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals (GB / T 35892-2018) and were approved by the Ethics Committee of the School of Basic Medical Sciences, Jilin University (Approval No.: 2024614). All procedures were performed in accordance with relevant guidelines and regulations.

[0086] Flu Vac: The antigen is an inactivated FM1 influenza virus with a hemagglutination titer of 1024 HAU / 200 µl, and the adjuvant is an oil-in-water emulsion. It was prepared by the Department of Immunology, School of Basic Medical Sciences, Jilin University.

[0087] Single-stranded deoxy oligonucleotides (ILOs) having the sequence (5'-CTTGTCACGCTCAGCCTCGATGAA-3') as described in Example 1.

[0088] The culture media used for cell culture were as follows: Complete RPMI medium: based on RPMI 1640 (Gibco, cat#C11875500BT), supplemented with 10% (v / v) fetal bovine serum (FBS) (Gibco, cat#C11875500BT), 100 IU / mL penicillin and 100 IU / mL streptomycin (Gibco, cat#15140148); Serum-free RPMI medium: without FBS, containing 100 IU / mL penicillin and 100 IU / mL streptomycin; RPMI medium with 2% FBS: containing 2% (v / v) FBS, 100 IU / mL penicillin and 100 IU / mL streptomycin.

[0089] PBS (Biopico, China).

[0090] Antibodies used for flow cytometry analysis: CD3-APC (BD, Cat#581042), CD4-FITC (BD, Cat#553046), CD274-PE (Invitrogen, Cat#2284281).

[0091] 4.2. Methods

[0092] like Figure 3 As shown, BALB / c mice were subcutaneously injected with 100 μL of inactivated FM1 influenza virus vaccine (FluVac) on the medial aspect of the right thigh (near the inguinal lymph node region). Forty-eight hours later, 100 μL of PBS solution containing ILO (10 μg per mouse) was injected at the same injection site. Twenty-four hours later, ILN cells (ILNCs) were isolated from the ipsilateral inguinal lymph nodes (ILNs). Figure 3 A). In another independent experiment, BALB / c mice were administered according to ( Figure 3 A) The mice were inoculated with a mixture of FluVac and ILO (10 μg per mouse) as described above. Forty-eight hours later, ALN cells (ALNCs) were isolated from ipsilateral axillary lymph nodes (ALNs). Figure 3F). Collected ILNs or ALNs were placed in a culture dish containing 1 mL of ice-cold RPMI-1640 (supplemented with 10% FBS) and gently ground into a single-cell suspension using a ground glass slide. The cell suspension was filtered through a 300-mesh filter into a 1.5 mL Eppendorf tube and centrifuged at 3000 rpm for 5 minutes. After discarding the supernatant, the cell pellet was resuspended in 50 μL of PBS (Biopico, China) containing 2% FBS. The following antibodies, diluted 1:10 with PBS, were added: CD3-APC (BD, Cat#581042), CD4-FITC (BD, Cat#553046), and CD274-PE (Invitrogen, Cat#2284281). Cells were incubated on ice for 30 minutes, washed with PBS, centrifuged at 3000 rpm for 5 minutes, and finally resuspended in 200 μL of PBS. An isotype control was included to assess non-specific binding. Finally, cells were analyzed using flow cytometry (BD Biosciences, Accuri C6 Plus, RRID: SCR_019591), and raw data were processed using BD Accuri C6 Plus software version 1.0.23.1. Live cells were phylogenetically selected as the P1 population. PD-L1 expression levels were characterized by mean fluorescence intensity (MFI). The analysis included total ILNCs (…). Figure 3 C) or ALNCs Figure 3 The proportion and PD-L1 expression level of PD-L1⁺ cells (cells expressing PD-L1) in H); the proportion and PD-L1 expression level of PD-L1⁺CD3⁺ cells (cells co-expressing PD-L1 and CD3) in CD3⁺ cells. Figure 3 D, I); the proportion of PD-L1-CD4-cells (cells co-expressing PD-L1 and CD4) and the PD-L1 expression level on CD4-cells ( Figure 3 E, J). Data are expressed as mean ± standard deviation. Unpaired t-tests were used to assess statistical significance. The symbols * and ** indicate p < 0.05 and p < 0.01, respectively.

[0093] 4.3. Results

[0094] like Figure 3 As shown, ILO can inhibit the expression of PD-L1 in draining lymph node cells (ILNCs and ALNCs) of influenza virus vaccine-immunized mice. Figure 3 C, Figure 3 H); ILO can inhibit CD3⁺ cells in draining lymph nodes (ILNCs and ALNCs) of mice immunized with influenza virus vaccine. Figure 3 D, I) and CD4⁺ cells ( Figure 3 PD-L1 expression levels on E and J.

[0095] These results ( Figure 3 This study indicates that ILO can serve as an adjuvant for influenza vaccines, enhancing vaccine efficacy by inhibiting PD-L1 expression on T lymphocytes. The interaction between PD-1 and PD-L1 transmits inhibitory signals, leading to T cell exhaustion and weakening their effector function. Following vaccination, PD-1 expression is upregulated in vivo. Blocking PD-L1 significantly enhances vaccine efficacy by relieving T cell inhibition, maintaining a robust antiviral T cell response, and enhancing the activation of CD4⁺ and CD8⁺ T cells. After vaccination, activated antigen-presenting cells and T cells express PD-L1, which binds to PD-1 on T cells, transmitting inhibitory signals that lead to T cell exhaustion and weaken the formation of immune memory. By using anti-PD-L1 blocking antibodies, this "braking" signal can be removed, thereby restoring and enhancing the proliferative capacity, cytokine (such as IFN-γ) secretion function, and cytotoxic activity of vaccine-specific T cells, promoting stronger effector immunity and the establishment of long-lasting immune memory. Therefore, using PD-L1 blockade as an immune adjuvant is a cutting-edge strategy to improve the protective efficacy of existing vaccines [Mol Immunol. 2025 Dec;188:98-110; Vaccines (Basel). 2023 Mar 1;11(3):559].

[0096] These results ( Figure 3 This also indicates that ILO can enhance anti-tumor T-cell immunity by downregulating PD-L1 expression on T lymphocytes. Its core mechanism lies in directly relieving the functional inhibition of T cells themselves. PD-L1 expression on the surface of activated T cells binds to PD-1 receptors on their own or surrounding cells, transmitting inhibitory signals, leading to inhibited T-cell proliferation and decreased effector functions (such as cytokine secretion and cytotoxicity), i.e., entering a state of functional exhaustion. ILO, by specifically reducing PD-L1 levels on T cells, weakens the signal input of this inhibitory pathway from within the T cell, enabling T cells to maintain a stronger activated state, proliferative capacity, and killing function. Therefore, even without external antibody blockade, directly downregulating PD-L1 expression on T cells can effectively enhance the anti-tumor immune response of T cells.

[0097] These results ( Figure 3This study also demonstrated that ILO, by downregulating PD-L1 expression on T lymphocytes, possesses the potential to serve as a highly effective adjuvant for personalized neoantigen vaccines. Its core mechanism lies in ILO's ability to precisely remove the key intrinsic inhibition encountered by specific T cells induced by tumor vaccines during the response. Personalized tumor vaccines aim to activate tumor-specific T-cell immunity by delivering patient-specific neoantigens. However, activated T cells express PD-1, and under tumor microenvironment or continuous antigen stimulation, PD-L1 expression is upregulated on both the T cells themselves and on antigen-presenting cells. This upregulation, via the PD-1 / PD-L1 pathway, feedback-inhibits T-cell function, leading to rapid depletion and severely weakening the long-term protective effect of the vaccine. The unique value of ILO as an adjuvant lies in its ability to not merely non-specifically enhance immune activation, but rather specifically target and downregulate PD-L1 expression on those T cells activated by the vaccine. This effect is spatiotemporally synchronized with the vaccine-induced immune response. When a vaccine activates neoantigen-specific T cells and triggers PD-L1 upregulation, ILO can intervene in a timely manner to block the generation of this inhibitory signal from inside the cell.

[0098] Example 5: Inhibitory effect of CpG motif-containing interfering PD-L1 expression-enhancing deoxy oligonucleotides (ILOs) on tumor growth in melanoma-bearing mice.

[0099] 5.1. Materials

[0100] Six- to eight-week-old C57BL / 6J mice (RRID: IMSR_JAX:000664) were purchased from Changchun E-S Laboratory Animal Technology Co., Ltd., China. These mice were housed under specific pathogen-free (SPF) conditions at the Laboratory Animal Center of Jilin University, with free access to food and water. All mouse experiments were conducted in accordance with the Chinese Guidelines for Ethical Review of Laboratory Animal Welfare (GB / T 35892-2018), and were approved by the Ethics Committee of the School of Basic Medical Sciences, Jilin University (Approval No.: 2024614). All procedures were performed in accordance with relevant guidelines and regulations.

[0101] B16 melanoma cells (B16 cells; RRID: CVCL_0158) (purchased from the American Type Culture Collection) were cultured in RPMI-1640 medium (Gibco, catalog number C11875500BT) supplemented with 10% heat-inactivated fetal bovine serum (FBS; OriCell, catalog number FBSST-01033-500) and antibiotics (penicillin and streptomycin, 100 IU / mL each; Gibco, catalog number 15140148). Cells were maintained in a humidified environment at 37°C and 5% CO2, and were used within 2 months after resuscitation, with a passage count not exceeding 20 times. The B16 cells were maintained by the Department of Immunology and Biology, Jilin University, and were regularly screened for mycoplasma contamination using a mycoplasma detection kit (Yeasen Biotech, catalog number 40612ES25), with consistently negative results.

[0102] Single-stranded deoxy oligonucleotides (ILOs) having the sequence (5'-CTTGTCACGCTCAGCCTCGATGAA-3') as described in Example 1.

[0103] PBS (Biopico, China).

[0104] 5.2. Methods

[0105] like Figure 4 As shown, B16 cells (1×10⁻⁶) were used. 5 10 mice (n=10 per group) were subcutaneously injected into the right medial thigh (near the inguinal lymph node drainage area) of C57BL / 6J mice. ILO (20µg / mouse) or PBS was administered at the injection site on days 1, 3, 5, 7, and 9 post-inoculation. Figure 4 A). Record tumor growth (monitored via volume measurement) and survival. Figure 4 B) Tumor growth curves of mice treated with PBS (left) or ILO (right). Notably, seven out of ten mice treated with ILO did not develop palpable tumors. Figure 4 C) Mean tumor growth curves in mice treated with PBS or ILO. Figure 4 D) Survival curves of mice. Tumor volume data are presented as individual growth curves ( Figure 4 B) or mean ± standard error ( Figure 4 C) represents the tumor volume (mm³). The formula for calculating tumor volume (mm³) is: length (longest diameter, mm) × width² (shortest diameter, mm) × 0.52. Two-way ANOVA was used to compare tumor growth, and Mantel–Cox log-rank test was used for survival analysis. **p<0.01.

[0106] 5.3 Results

[0107] The results showed that ILO treatment significantly inhibited tumor growth ( Figure 4 B, C), significantly prolonged the survival of mice bearing melanoma ( Figure 4 D). This demonstrates that the anti-tumor deoxy oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression provided by this invention can treat melanoma.

[0108] Example 6: Antitumor deoxygenated oligonucleotides (ILOs) containing CpG motifs that interfere with PD-L1 expression exert their inhibitory effect on mouse lung cancer cell growth by synergistically activating TLR9 and reducing PD-L1 expression.

[0109] 6.1 Materials

[0110] Six- to eight-week-old C57BL / 6J mice (RRID: IMSR_JAX:000664) were purchased from Changchun E-S Laboratory Animal Technology Co., Ltd., China. These mice were housed under specific pathogen-free (SPF) conditions at the Laboratory Animal Center of Jilin University, with free access to food and water. All mouse experiments were conducted in accordance with the Chinese Guidelines for Ethical Review of Laboratory Animal Welfare (GB / T 35892-2018), and were approved by the Ethics Committee of the School of Basic Medical Sciences, Jilin University (Approval No.: 2024614). All procedures were performed in accordance with relevant guidelines and regulations.

[0111] Lewis lung cancer (LLC) cells (purchased from the U.S. Type Culture Collection) were cultured in RPMI-1640 medium (Gibco, catalog number C11875500BT) supplemented with 10% heat-inactivated fetal bovine serum (FBS; OriCell, catalog number FBSST-01033-500) and antibiotics (penicillin and streptomycin, 100 IU / mL each; Gibco, catalog number 15140148). LLC cells were cultured in a humidified environment at 37°C and 5% CO2, with a post-resuscitation period not exceeding 2 months and a passage count not exceeding 20 times. This cell line was maintained by the Department of Immunology, Jilin University, and was regularly screened for mycoplasma contamination using a mycoplasma detection kit (Yeasen Biotech, catalog number 40612ES25), with consistently negative results.

[0112] Examples of ILO (5'-CTTGTCACGCTCAGCCTCGATGAA-3') (an antitumor deoxygenated oligonucleotide containing a CpG motif that interferes with PD-L1 expression), No-CpG-ILO (5'-CTTGTCAGCCTCAGCCCCCATGAA-3') (a control oligonucleotide that retains the common ILO sequence but lacks the CpG motif), and No-ASO-ILO (5'-GAACAGTGCGAGTCGGAGCTACTT-3') (an ILO sense control oligonucleotide that retains the CpG motif) are shown in Example 1.

[0113] PBS (Biopico, China)

[0114] 6.2 Methods

[0115] like Figure 5 As shown, on day 0, Lewis lung cancer cells (1 × 10⁶ cells per mouse) were subcutaneously inoculated into the inner right thigh (n=10 per group) of C57BL / 6 mice (n=10 per group). 5 (cells). Subsequently, on days 1, 3, 5, 7, and 9, mice were injected peritumorally at the inoculation site with ILO (20 µg / mouse), No-ASO-ILO, No-CpG-ILO, or PBS, respectively. Tumor growth was then continuously monitored. Figure 5 B) Individuals and ( Figure 5 C) Individual mean tumor growth curve. Tumor volume was calculated using the formula: Volume (mm³) = Major diameter (longest diameter, mm) × [Minor diameter (shortest diameter, mm)]² × 0.52. Data are presented as individual growth curves or mean ± standard error (mean ± SEM). Tumor volume comparisons were performed using two-way ANOVA, and survival analysis was performed using the Mantel-Cox log-rank test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0116] 6.3 Results

[0117] like Figure 5As shown, the antitumor deoxygenated oligonucleotide (ILO) containing a CpG motif that interferes with PD-L1 expression, provided by this invention, exhibits the most significant inhibitory effect on lung cancer cell growth. Its mechanism of action is based on its dual function as a TLR9 agonist and a PD-L1 antisense oligonucleotide, allowing it to synergistically exert its effects by simultaneously enhancing immune attack and relieving tumor immunosuppression. On one hand, the CpG motif in the ILO molecule acts as a TLR9 agonist, which can be recognized by TLR9 on the surface of immune cells (such as dendritic cells and B cells) in the tumor microenvironment, activating the downstream MyD88-dependent signaling pathway, inducing the production of pro-inflammatory cytokines (such as IL-12 and IFN-α) and the maturation of antigen-presenting cells, thereby promoting the activation and functional enhancement of effector cells such as NK cells and CD8⁺ T cells, and comprehensively improving the strength of the antitumor immune response. On the other hand, the antisense oligonucleotide sequence contained in the ILO can specifically target PD-L1 mRNA in tumor cells, effectively downregulating PD-L1 protein expression at the post-transcriptional level through steric hindrance or RNase H-mediated degradation mechanisms. This effect directly weakens the inhibition of T cell function by tumor cells through the PD-L1 / PD-1 pathway, while also alleviating the immunosuppressive state mediated by immunosuppressive cells in the tumor microenvironment. The dual functional modules exhibit a synergistic effect: TLR9 activation promotes the recruitment and activation of immune cells, creating a microenvironment conducive to immune attack; while PD-L1 downregulation removes key inhibitory signals encountered by effector cells in recognizing and killing tumor cells. This synergistic effect of "enhancing immune attack" and "relieving immunosuppression" allows ILO to more effectively reshape the tumor immune microenvironment, thereby achieving the strongest inhibitory effect on lung cancer cell growth.

[0118] Example 7 Effects of CpG-Modified Interfering PD-L1 Antitumor Deoxyoligonucleotides (ILOs) on Lewis Lung Cancer Cell-Forming Tumor Tissue and Draining Lymph Node Cells in Mice

[0119] 7.1 Materials

[0120] Six- to eight-week-old C57BL / 6J mice (RRID: IMSR_JAX:000664) were purchased from Changchun E-S Laboratory Animal Technology Co., Ltd., China. These mice were housed under specific pathogen-free (SPF) conditions at the Laboratory Animal Center of Jilin University, with free access to food and water. All mouse experiments were conducted in accordance with the Chinese Guidelines for Ethical Review of Laboratory Animal Welfare (GB / T 35892-2018), and were approved by the Ethics Committee of the School of Basic Medical Sciences, Jilin University (Approval No.: 2024614). All procedures were performed in accordance with relevant guidelines and regulations.

[0121] Lewis lung cancer (LLC) cells (purchased from the U.S. Type Culture Collection) were cultured in RPMI-1640 medium (Gibco, catalog number C11875500BT) supplemented with 10% heat-inactivated fetal bovine serum (FBS; OriCell, catalog number FBSST-01033-500) and antibiotics (penicillin and streptomycin, 100 IU / mL each; Gibco, catalog number 15140148). LLC cells were cultured in a humidified environment at 37°C and 5% CO2, with a post-resuscitation period not exceeding 2 months and a passage count not exceeding 20 times. This cell line was maintained by the Department of Immunology, Jilin University, and mycoplasma contamination was regularly screened using a mycoplasma detection kit (Yeasen Biotech, catalog number 40612ES25), with consistently negative results. LLC cells were cultured in complete RPMI-1640 medium at 37°C and 5% CO2, and mycoplasma testing was performed regularly, with all results being negative.

[0122] Examples of ILO (5'-CTTGTCACGCTCAGCCTCGATGAA-3') (an antitumor deoxygenated oligonucleotide containing a CpG motif that interferes with PD-L1 expression), No-CpG-ILO (5'-CTTGTCAGCCTCAGCCCCCATGAA-3') (a control oligonucleotide that retains the common ILO sequence but lacks the CpG motif), and No-ASO-ILO (5'-GAACAGTGCGAGTCGGAGCTACTT-3') (an ILO sense control oligonucleotide that retains the CpG motif) are shown in Example 1.

[0123] PBS (Biopico, China)

[0124] Antibodies used for flow cytometry analysis: CD3-APC (BD, Cat#581042), CD4-FITC (BD, Cat#553046), CD8-PE (BD, Cat#553033), CD274-PE (Invitrogen, Cat#2284281), Foxp3-APC (BD, 560401, RRID: AB_1645201).

[0125] 7.2 Methods

[0126] like Figure 5 As shown in Figure A, on day 0, Lewis lung cancer (LLC) cells (1×10⁻⁶) were subcutaneously inoculated into the right medial thigh near the inguinal lymph node drainage area of ​​C57BL / 6J mice (n=9 per group). 5 Mice were injected with ILO (20 µg / mouse), No-ASO-ILO (20 µg / mouse), No-CpG-ILO (20 µg / mouse), or PBS at the inoculation site on days 1, 3, 5, 7, and 9, respectively. Mice were sacrificed on day 25 post-tumor inoculation. Tumor tissue was collected. Figure 6 A, B, C) and tumor draining lymph nodes (tDLNs) Figure 6 (D, E). tDLNs were inguinal lymph nodes. Collected tumor tissue or tDLNs were placed in a culture dish containing 1 mL of ice-cold RPMI-1640 (supplemented with 10% FBS) and gently ground into a single-cell suspension using a ground glass slide. The cell suspension was filtered through a 300-mesh filter into a 1.5 mL Eppendorf tube and centrifuged at 3000 rpm for 5 minutes. After discarding the supernatant, cells from tumor tissue or tDLNs were resuspended in 50 µL of PBS (Biopico, China) containing 2% FBS. Cells were stained using the following antibodies diluted 1:10 in PBS: PD-L1 (CD274-PE), Foxp3-APC (for tumor-derived cells only); CD8-PE and CD4-FITC. Foxp3-APC was used for intracellular staining. Isotype controls were set up to assess non-specific binding. Finally, cells were analyzed using flow cytometry (BD Biosciences, Accuri C6 Plus, RRID: SCR_019591), and raw data were processed using BD Accuri C6 Plus software version 1.0.23.1. Cells isolated from tumor tissue were delineated as population P1 (…). Figure 6 A), further divided into P2 group (total tumor tissue cells) and P3 group (tumor cells) Figure 6C). Cells isolated from tDLNs were delineated as population P4 (D). PD-L1 expression levels were expressed as mean fluorescence intensity (MFI). The analysis included: the percentage of FOXP-3⁺CD4⁺, CD4⁺, and CD8⁺ cells in total tumor tissue cells (B); and the expression levels of PD-L1 in total tumor tissue cells (including tumor cells and tumor-infiltrating T lymphocytes) and on tumor cells (C). Figure 6 C); Percentage of CD4⁺ and CD8⁺ cells in tDLNs ( Figure 6 (D) Expression levels of PD-L1 on CD4⁺ and CD8⁺ T cell subsets within tDLNs (E). Data are expressed as mean ± standard deviation. Statistical significance was determined using unpaired t-tests, *p < 0.05, **p < 0.01.

[0127] 7.3 Results

[0128] like Figure 6 As shown, ILO treatment synergistically remodels the immune microenvironment of mouse tumor tissue, which is an important mechanism for its highly effective anti-tumor effect. On the one hand, the number and composition of immune cells in tumor tissue are significantly altered. The number of CD4⁺ (helper T cells) and CD8⁺ (cytotoxic T cells) increases significantly, indicating enhanced recruitment and expansion of effector immune cells at the tumor site. At the same time, the number of FOXP-3⁺CD4⁺ Treg cells with immunosuppressive function is significantly reduced, suggesting that the immunosuppressive state in the tumor is alleviated. On the other hand, PD-L1 expression is widely suppressed in both tumor cells and tumor-infiltrating immune cells (including CD4⁺ T cells, CD8⁺ T cells, and Treg cells). This widespread downregulation has a dual effect: at the tumor cell level, it directly weakens the immune escape ability mediated by the PD-L1 / PD-1 pathway; at the immune cell level, the reduction in T cell PD-L1 expression may reduce its functional suppression and help maintain its activated state. In summary, ILO systematically reshapes the tumor immune microenvironment by simultaneously regulating the composition of immune cells (increasing effector cells and reducing suppressor cells) and inhibiting the expression of the key immune checkpoint molecule PD-L1, thereby synergistically promoting a potent and sustained anti-tumor immune response.

Claims

1. A CpG motif-containing antitumor deoxy oligonucleotide that interferes with PD-L1 expression, characterized in that: The single-stranded deoxy oligonucleotide with a fully thiolated backbone as shown in SEQ ID NO: 1 is named ILO.

2. The antitumor deoxy oligonucleotide containing a CpG motif that interferes with PD-L1 expression according to claim 1, which may be chemically modified.

3. The antitumor deoxygenated oligonucleotide containing a CpG motif that interferes with PD-L1 expression according to claims 1 and 2, which can exert antitumor effects by activating antigen-presenting cells and reducing the expression of PD-L1 protein in tumor cells and immune cells.

4. The antitumor deoxy oligonucleotide containing a CpG motif that interferes with PD-L1 expression according to claims 1 and 2 can be used alone or in combination with antitumor agents for individual tumor treatment.

5. The antitumor deoxy oligonucleotide containing a CpG motif that interferes with PD-L1 expression according to claims 1 and 2 can be used alone or in combination with antitumor agents for individual treatment of lung cancer.

6. The antitumor deoxy oligonucleotide containing a CpG motif that interferes with PD-L1 expression according to claims 1 and 2 can be used alone or in combination with antitumor agents for individual treatment of melanoma.

7. The antitumor deoxygenated oligonucleotide containing a CpG motif that interferes with PD-L1 expression, as described in claims 1 and 2, can enhance the efficacy of influenza virus vaccines.

8. The antitumor deoxy oligonucleotide containing a CpG motif that interferes with PD-L1 expression according to claims 1 and 2 can be delivered via an antitumor drug delivery system to treat tumors.

9. The antitumor deoxy oligonucleotide containing a CpG motif that interferes with PD-L1 expression according to claims 1 and 2 can be used to treat tumors via systemic or local administration.

10. The antitumor deoxygenated oligonucleotide containing a CpG motif that interferes with PD-L1 expression according to claims 1 and 2, which does not induce complement-dependent cytotoxicity or antibody-dependent cell-mediated cytotoxicity caused by immune checkpoint antibodies.

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