Bifunctional oligonucleotide having antitumor effect

Abstract

The present invention provides two bifunctional single-stranded deoxyoligonucleotides with specific sequences. They can be used alone or in combination with other anti-tumor agents to treat tumors, and can also be used as adjuvants for tumor vaccines.

Description

Technical fields:

[0001] This invention relates to bifunctional single-stranded deoxy oligonucleotides with antitumor activity. These oligonucleotides have specific sequences and can be used alone or in combination with other antitumor agents to treat tumors, and can also be used as adjuvants for tumor vaccines. Background technology:

[0002] Single-stranded deoxyoligodeoxynucleotides with specific base sequences can activate an individual's innate immune responses and adaptive immune responses to tumor cells, thus exhibiting anti-tumor effects [Akira S, et al. Nature. 2000; 408: 740-745; Klinman DM. Nat Rev Immunol. 2004; 4: 249-259; Krieg AM, et al. J Clin Invest. 2007; 117: 1184-94; Scheiermann J, et al. Vaccine. 2014 Nov 12; 32(48): 6377-89].Due to the aforementioned activities, single-stranded deoxy oligonucleotides with specific base sequences may be used alone or in combination with other antitumor agents to treat tumors, and may also be used as adjuvants to tumor vaccines to enhance their efficacy [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: 73946; VaImori D, et al. Proc Natl Acad Sci US A. 2007; 104: 8947-52; Carpentier A, et al. Neuro Oncol. 2010; 12: 401-8; Hirsh V. et al. J Clin Oncol. 2011; 29: 2667-2674; Smith DA, et al. Cancer Immunol Immunother. 2014; 63: 787-796; Chan E. et al. Cancer Chemother Pharmacol. 2015; 75: 701-9; Friedbeag JW, et al. Br J Haematol. 2009; 146: 282-91; Brody JD, et al. Blood. 2009; 113: 85-94; Lim SH, et al. J Immunol Ref. 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. J Immunother. 2006; 29:: 558-68; Reilley MJ, et al, J Immunother Cancer.2019 Nov 26;7(1):323; Mangsbo SM, et al. al. IntImmunol. 2010;22:651-60].

[0003] T lymphocytes (T cells) are key cells in the adaptive immune system that play a crucial role in anti-tumor activity. T cells include cytotoxic T cells (CD8+). + T cells and helper T cells (CD4+) + T cells). CD8 + T cells directly recognize and kill tumor cells. CD4 + T-cell-assisted CD8 + T cells kill tumor cells or directly kill tumor cells [Sharma P, et al. Cell. 2023 Apr 13; 186(8): 1652-1669]. T cells can only kill tumor cells after they are activated. T cell activation requires two signals. The first signal is generated by the T cell receptor (TCR) recognizing and binding to the MHC-antigen peptide complex. CD8 + T cells recognize and bind to complexes of MHC class I molecules and tumor antigens on the surface of tumor cells. CD4 + T cells recognize and bind to complexes of MHC class II molecules and tumor antigens on the surface of antigen-presenting cells. A second signal is generated through the interaction of co-stimulatory molecules such as CD28 and CD86. CD86 is expressed on the surface of antigen-presenting cells or tumor cells. CD28 is expressed on the surface of T cells [Sharma P, et al. Cell. 2023 Apr 13; 186(8): 1652-1669]. Upon activation by the dual signal, CD8... + T cells and CID4 + T cells upregulate CTLA-4 expression. CTLA-4 is cytotoxic T-lymphocyte associated protein 4, expressed on the surface of T lymphocytes. CTLA-4 is a co-inhibitory molecule that competitively binds to CD86 on the surface of antigen-presenting cells, along with CD28 on the surface of T cells. This binding initiates an inhibitory signaling pathway, inhibiting CD4+. + T cells and CD8 + T cell activation, thus inhibiting CD8 + T cells and CD4 + T cells kill tumor cells [Sharma P. et al. Scieace. 2015 Apr 3; 348(6230): 56-61; Greenwald RJ, et al. Annu RevImmunol. 2005; 23: 515-48; Sharma P, et al. Cell. 2023 Apr 13; 186(8): 1652-1669]. Blocking or inhibiting the interaction between CTLA-4 and CD86 molecules can enhance the killing of tumor cells by anti-tumor T cells (CD4+). +T cells and CD8 + T cells are in a state of continuous and sufficient activation to kill tumor cells. Blocking or inhibiting the inhibitory signaling pathway initiated by CTLA-4 can enhance the individual's anti-tumor cell immune response. Preparations that block or inhibit the inhibitory signaling pathway initiated by CTLA-4 can become tumor therapeutic drugs [Sharma P. et al. Science. 2015 Apr 3; 348(6230): 56-61; Greenwald RJ, et al. Annu Rev Immumol. 2005; 23: 515-48; Sharma P, et al. Cell. 2023 Apr 13; 186(8): 1652-1669].

[0004] Preclinical studies have demonstrated that antibodies targeting CTLA-4 can treat a variety of tumors by blocking / inactivating the inhibitory signaling pathway initiated by CTLA-4. These tumors include breast cancer [Hurwitz AA, et al. Proc Natl Acad SciUSA. 1998 Au8 18; 95(17): 10067-71], melanoma [van Elsas A., et al. J Exp Med. 1999 Aug 2; 190(3): 355-66: Hodi, FSet al. N Engl J Med. 2010 Aug 19; 363(8): 711-23], and prostate cancer [Waitz R, et al. Oncoimmunology. 2012 Jul 1; 1(4): 544-546: van den Eertwegh AJ, et al., Lancet Oncol. 2012 May; 13(5): 509-17].

[0005] Clinical trials have shown that the antibody targeting CTLA-4 (Ipilimumab) is effective against melanoma [Hodi FS. et al., NEn81 J Med. 2010 Aug 19; 363(8): 711-23], renal cell carcinoma [Yang JC et al., J Immunother. 2007 Nov-Dec; 30(8): 825-30], prostate cancer [van den Eertwegh AJ. et al., Lancet Oncol. 2012 May; 13(5): 509-17], bladder cancer [Carthon BC et al., Clin Cancer Res. 2010 May 15; 16(10): 2861-71], and ovarian cancer [Hodi FS. et al., Proc Natl Acad Sci U SA. 2008 Feb]. 26; 105(8): 3005.10] and non-Hodgkin's lymphoma [Ansell SM., et al. Clin Cancer Res. 2009 Oct 15; 15(20): 6446-531]. In 2011, Ipilimumab (humanized CTLA-4 monoclonal antibody) was approved by the U.S. Food and Drug Administration (FDA) for the treatment of melanoma [Sharma P, et al. Nat Rev Cancer. 2011 Oct 24; 11(11): 805-12; Pardoll DM, Nat Rev Cancer. 2012 Mar 22; 12(4): 252-64], and was later approved for the treatment of colon cancer and renal cell carcinoma [Ramos-Casals M, et al. 2020 May 7; 6(1): 38]. 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 [ShoT, et al. Taaget Oncol. 2024 Sep; 19(5): 76%778].

[0006] CTLA-4 antibodies (Ipilimumab and Tremelimumab) belong to the immune checkpoint inhibitors (ICIs). Other approved ICIs for cancer treatment include PD-1 and PD-L1 antibodies.

[0007] Cancer therapeutic ICIs, including CTLA-4 antibodies, can trigger immune-related adverse events (irAEs). irAEs are a variety of diseases caused by the use of cancer therapeutic ICIs, affecting almost any organ system and potentially life-threatening [Puzanov I, et al. J ImmunotherCancer. 2017 Nov 21; 5(1): 95]. The prevalence and severity of irAEs underscore the necessity of developing “non-antibody” cancer therapeutic ICIs. Summary of the Invention:

[0008] 1. This invention provides two bifunctional single-stranded deoxy oligonucleotides (hereinafter referred to as bifunctional oligonucleotides), the sequences of which are shown in the sequence listing. <400> 1 and sequence list <400> As shown in Figure 2, these two bifunctional oligonucleotides are named ICO-2 and ICO-3, respectively. ICO-2 is a sequence listed as shown in the sequence table. <400> ICO-3 is a bifunctional oligonucleotide with the sequence shown in Figure 1. <400> The bifunctional oligonucleotide sequence shown in Figure 2.

[0009] 2. ICO-2 and ICO-3 can be chemically modified or restructured.

[0010] 3. ICO-2 and ICO-3 can activate the innate immune response and interfere with the translation of proteins using CTLA4 mRNA as a template, making them bifunctional activators and inhibitors.

[0011] 4. ICO-2 and ICO-3 enhance an individual's innate and adaptive immune responses to tumor cells.

[0012] 5. When applied to individuals, ICO-2 and ICO-3 promote the killing of tumor cells, inhibit tumor formation, and prolong the survival of tumor-bearing individuals, demonstrating a tumor treatment effect.

[0013] 6. ICO-2 and ICO-3 can be used alone or in combination with other antitumor agents to treat tumors.

[0014] 7. ICO-2 and ICO-3 can be used as adjuvants for tumor vaccines.

[0015] Terminology in the invention:

[0016] 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.

[0017] Oligonucleotides: Oligonucleotides are molecules composed of multiple nucleotides, ranging from a few to dozens. Nucleotides are the basic building blocks of nucleic acids and oligonucleotides. Nucleotides consist of nucleosides and phosphate groups. Nucleosides consist of pentose sugars and bases. Pentoses include ribose and deoxyribose. Pentoses and bases are linked to form nucleosides. Nucleosides are linked by phosphate groups to form nucleotides. Nucleotides are linked by phosphodiester bonds to form oligonucleotides. The bases that make up nucleosides include pyrimidines and purines. Pyrimidines include thymine (T or t) and cytosine (C or c). Purines include adenine (A or a) and guanine (G or g). Bases in oligonucleotides can be rare bases. Rare bases include, but are not limited to, 5-hydroxymethylcytosine, 7-methylguanine, and 5-hydroxymethylcytosine. Oligonucleotides can be single-stranded, double-stranded, cyclic, or have a cyclic structure. In this invention, oligonucleotide (ODN) can be represented by its abbreviation ODN. The sequence of nucleotides in an oligonucleotide constitutes its primary structure, also known as the nucleotide sequence. The nucleotide sequence can be represented by a base sequence; therefore, the sequence of a nucleotide is also called a base sequence. The sequence of deoxyoligonucleotides can be represented by the abbreviations of bases: T or t represents thymine, C or c represents cytosine, A or a represents adenine, and G or g represents guanine.

[0018] "Oligonucleotides provided by this invention": The oligonucleotides provided by this invention are two bifunctional single-stranded deoxy oligonucleotides, also known as bifunctional oligonucleotides, and their sequences are shown in the sequence listing. <400> 1 and sequence list <400> As shown in Figure 2, these two bifunctional oligonucleotides are named ICO-2 and ICO-3, respectively. ICO-2 is a sequence listed as shown in the sequence table. <400> ICO-3 is a single-stranded deoxy oligonucleotide with the sequence shown in Figure 1. <400> The sequence shown in Figure 2 is a single-stranded deoxy oligonucleotide.

[0019] "Chemical Modification": Compared to natural DNA, the bifunctional oligonucleotides (ICO-2 and ICO-3) provided by this invention can be chemically modified. Chemical modification is the phenomenon or method of altering the covalent structure by introducing or removing any chemical group. The chemical modification sites of ICO-2 and ICO-3 can occur at the phosphodiester bond, ribose, and bases. Chemical modification of ICO-2 and ICO-3 can occur at the 5' or 3' end, and can be performed during or after synthesis. This invention relates to the chemical modification of ICO-2 and ICO-3, including but not limited to modifications of the oligonucleotide backbone, such as thiomodification (replacing the non-bridging oxygen atom of the phosphate group in the internucleotide phosphodiester bond with a sulfur atom) and substitution modification (including substitution of alkyl, aromatic, or any other chemical group). Chemical modification of ICO-2 and ICO-3 also includes base substitution and base modification, where the substituted base can be a rare base or a derivative of various bases. Chemical modification of ICO-2 and ICO-3 also includes attaching one or more nucleotides and / or any other chemical group to their 5' and / or 3' ends.

[0020] "Immune Response": The bifunctional oligonucleotides (ICO-2 and ICO-3) provided by this invention can be used to treat individual tumors by enhancing the immune response to tumor cells and tumor antigens. Immune response and immune reaction have the same meaning. Immune response is divided into innate immune response and adaptive immune response. An immune response to tumor cells can limit tumor cell growth and eliminate tumor cells. Promoting an individual's immune response to tumor cells can produce an anti-tumor effect.

[0021] "Individual": In this invention, "subject" refers to humans and non-human vertebrates.

[0022] "Tumor": In this invention, the terms "tumor" and "cancer" are used interchangeably. Tumors include solid tumors, soft tissue sarcomas, and myeloid or lymphoid system tumors. The bifunctional oligonucleotides (ICO-2 and ICO-3) provided by this invention are used alone or in combination with other antitumor agents to treat tumors. These tumors include, but are not limited to, melanoma, colon cancer, and lung cancer.

[0023] "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 bifunctional oligonucleotides (ICO-2 and ICO-3) provided by this invention can be used for tumor treatment.

[0024] "Anti-tumor agent": An anti-tumor agent is a preparation that can treat tumors after being applied to an individual. In this patent, the terms "preparation" and "drug" are interchangeable; anti-tumor agents and anti-tumor drugs are also interchangeable. Anti-tumor agents include, but are not limited to, chemotherapy drugs, tumor vaccines, immune checkpoint inhibitory antibodies, oncolytic viruses, tumor angiogenesis inhibitors, tumor therapeutic antibodies, small molecule kinase inhibitors, and tumor therapeutic cells including dendritic cells, CAR-T cells, tumor-infiltrating lymphocytes, and natural killer cells. The bifunctional oligonucleotides (ICO-2 and ICO-3) provided by this invention may serve as anti-tumor agents, having a therapeutic effect on tumors occurring in an individual by enhancing the individual's anti-tumor immune response.

[0025] "Anti-tumor T-cell immune response": The anti-tumor T-cell immune response is an anti-tumor response mediated by T cells (T lymphocytes). T cells are the most potent anti-tumor cells in the adaptive immune system, including CD8+ cells. + T cells and CD4 + T-fine. CD8 + T cells are T cells that express the CD8 molecule on their surface and are cytotoxic T lymphocytes (CTLs). CD4 + T cells are T cells that express the CD4 molecule on their surface; they are helper T lymphocytes (Th). CD8 + T cells directly recognize and kill tumor cells. CD4 + T-cell-assisted CD8 + T cells kill tumor cells either directly or by secreting cytokines (tumor necrosis factor) [cao LL, et al. Immunity. 2023 oct 10; 56(10): 2206-2217]. The bifunctional oligonucleotides (ICO-2 and ICO-3) provided in this invention can produce anti-tumor effects by promoting and maintaining the activation of anti-tumor T cells.

[0026] "Dual Signals for T Cell Activation": Anti-tumor T cells can only kill tumor cells after being activated by tumor cells or tumor antigens. Activation of anti-tumor T cells requires two signals (a first signal and a second signal). The first signal is generated through the recognition and binding of the MHC-antigen peptide complex by the T cell antigen receptor (TCR). CD8 + T cells recognize and bind to complexes of MHC class I molecules and tumor antigens on the surface of tumor cells. CD4 +T cells recognize and bind to complexes of MHC class II molecules and tumor antigens on antigen-presenting cells (APCs). The second signal, also known as the co-stimulatory signal, is generated through the interaction between co-stimulatory molecules on the surface of APCs, including CD40, CD80, and CD86, and their corresponding ligand molecules (such as CD28) on the surface of T cells. Antitumor T cells that only receive the first activation signal cannot be fully activated and may even enter a state of immune tolerance. Only after receiving the first and second activation signals can antitumor T cells be fully activated and perform their antitumor function [Ramos-Casals M, et al. Nat Rev Dis Primers. 2020 May 7; 6(1): 38].

[0027] “Costimulatory molecules”: The costimulatory molecules involved in this invention are membrane proteins that enhance the signaling activity of the T cell receptor (TCR), including CD40, CD80, and CD86 [Cao LL, et al. Immunity. 2023 Oct 10; 56(10): 2206-2217]. CD86 is a costimulatory molecule expressed on antigen-presenting cells (APCs), including B lymphocytes and dendritic cells. During the immune response, costimulatory molecules interact with proteins on the surface of T cells, such as CD28, to provide a second signal for activating CD4+ T lymphocytes and CD8+ T lymphocytes. CD40 and CD86, expressed on the surface of B cells and dendritic cells, are costimulatory molecules that activate anti-tumor T cells. The bifunctional oligonucleotides (ICO-2 and ICO-3) provided by this invention can upregulate the expression of CD40 and CD86 on the surface of B cells and dendritic cells, thereby enhancing the second signal for anti-tumor T cell activation.

[0028] “Anti-tumor effect of B cells”: B cells assist in the activation of anti-tumor T cells, enabling them to exert a more efficient anti-tumor effect [Sagiv-Barfi I, et al. Sci Immunol. 2022 May 27; 7(71): eabn5859; Petitprez F, et al. Nature. 2020 Jan; 577(7791): 556.560; HeImink BA, et al. Nature. 2020 Jan; 577(7791): 549-555; Fridman WH, et al. NatRev Clin Oncol. 2022 Jul; 19(7): 441-457]. B cells present tumor antigens to T cells [Bruno TC, et al. Cancer Immunol Res. 2017 Octt; 5(10): 898-907. DiLillo DJ, et al. J Immunol. 2010 Apr 1; ​​184(7): 4006-16]. Blocking the antigen presentation function of B cells weakens the activation of tumor-specific T cells [Sagiv-Barfi I, et al. Sci Immunol. 2022 May 27; 7(71): eabn5859]. B cells express pattern recognition receptors (PRRs). B cells activated by PRRs can more effectively cooperate with T lymphocytes to kill tumor cells [Sagiv-Barfi I, et al. Intratumoral immunotherapy relies on B and T cell collaboration. Sci Immunol. 2022 May 27; 7(71): cabn5859].

[0029] Pattern recognition receptors (PRRs) are a class of structurally diverse proteins that share the common characteristic of recognizing microbial products (usually cell wall components or nucleic acids). When PRRs expressed by dendritic cells (DCs) detect microorganisms, they trigger various immunostimulatory activities, thereby promoting T cell-mediated immune responses. PRRs stimulate innate immune responses. PRRs include: Toll-like receptors (TLRs), RIG-I like receptors (RLRs), nucleotide binding domain leucine rich repeat containing proteins (NLRs), c-type lectin receptors (CLRs), and cyclic GMP-AMP synthase (cGAS) [Cao LL, et al. Immunity. 2023 Oct 10; 56(10): 2206-2217].

[0030] "Toll-like receptor-9 agonist": Toll-like receptor 9 (TLR9) is a PRR (pattern recognition receptor). B cells and dendritic cells express TLR9. CpG-containing oligonucleotides (CPG 0DN) are TLR9 agonists that can mimic pathogen DNA to activate B cells and dendritic cells, creating an anti-tumor immune microenvironment [Hemmi H, et al. Nature 408, 740-745 (2000); Celhar T, et al., Int. Immmol 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. Regulation of B-cell responses by Toll-like receptors. Immunology. 2012 Aug; 136(4): 370-9]. CPG ODN activates B cells and dendritic cells to upregulate co-stimulatory molecules, including CD80 and CD40, 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-BarfiI, et al. Sci Immunol. 2022 May 27; 7(71): eabn5859]. Preparations that can activate B cells and dendritic cells through TLR9 to upregulate CD80 and CD40 help activate anti-tumor T cells to kill tumor cells, thus having a tumor therapeutic effect. The bifunctional oligonucleotides (ICO-2 and ICO-3) provided by this invention contain CpG motifs and have the effect of CPG ODN to activate B cells and dendritic cells to upregulate the expression of CD80 and CD40, thus having anti-tumor efficacy.

[0031] CD19 + Lymphocytes: The CD19 molecule is a surface marker of B lymphocytes. Lymphocytes that express the CD19 molecule on their surface are B lymphocytes (B cells), also known as CD19 lymphocytes. + B lymphocytes or CD19 +Cells. CD19 is an inducible surface protein. Elevated CD19 expression levels indicate B cell activation [Sato S, et al. Quantitative genetic variation in CD19 expression correlates with autoimmunity. J Immunol. 2000 Dec1; 165(11): 6635-43]. CD19 + B lymphocytes express costimulatory molecules including CD40, CD80, and CD86. Elevated levels of CD80 and CD40 expression mark B cell activation. CpG ODN stimulates B cells to express CD80 and CD40. The bifunctional oligonucleotides (ICO-2 and ICO-3) provided by this invention can stimulate B lymphocytes to upregulate CD80 and CD40, exhibiting CpG ODN activity. ICO-2 and ICO-3 provided by this invention can increase specific CD19 in draining lymph nodes. + The number of lymphocytes.

[0032] CD11c + Dendritic cells: CD11c is a surface marker of dendritic cells (DCs). DCs expressing CD11c are called CD11c-expressing cells. + Dendritic cells. CD11c + Dendritic cells are antigen-presenting cells (APCs) that can present peptides of tumor antigens on their surface to activate antigen-specific T cells. CD11c +Dendritic cells express costimulatory molecules. The ICO-2 and ICO-3 provided in this invention can activate and increase the number of antigen-presenting cells (APCs) in draining lymph nodes. Increased APC numbers in draining lymph nodes promote the activation of antigen-specific T cells, thereby enhancing the immune response against tumor cells. The TLR signaling pathway in dendritic cells (DCs) can promote antigen capture and enhance antigen presentation on MHC-I and MHC-II molecules. Simultaneously, TLRs can upregulate T cell costimulatory molecules (including CD40, CD80, and CD86) [schnare M, et al. (2001) Nat Immunol 2, 947-950]. The costimulatory signal “CD40”: CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily and is widely expressed on the surface of various cell types, including B cells. The interaction between CD40 and its ligand (CD40L) can trigger a variety of immune responses, including granting dendritic cells the ability to activate CD8+ effector T cells and promoting the activation, proliferation and differentiation of B cells [Zhou Y, et al. Harnessing the potential of CD40 agonism in cancer therapy. Cytokine Growth Factor Rev. 2024 Feb; 75: 40-56].

[0033] “Co-inhibitory molecules”: Co-inhibitory molecules are membrane proteins that inhibit the signaling activity of T cell receptor (TCR), including CTLA-4 (cytotoxic T lymphocyte antigen 4) and PD-1 (programmed cell death protein 1) [Cao LL, et al. Immunity. 2023 Oct 10; 556(10): 2206-2217].

[0034] "Cytotoxic T-Lymphocyte Antigen 4": Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) is an inducible membrane protein expressed on activated CD4+. + T cells and CD8 + On the surface of T cells, CTLA-4 competitively binds to the co-stimulatory molecule CD28, which binds to CD80 and CD86 on antigen-presenting cells, transmitting inhibitory signals and limiting CD80 binding. + T cells and CD4 + T cell activation. This inhibitory signaling hinders CD8 activation. +T cells and CD4 + T cells kill tumor cells [Ramos-Casals M, et al. Nat Rev Dis Primers. 2020 May 7; 6(1): 38]. In the tumor microenvironment, CTLA-4-mediated T cell inhibitory signaling weakens the individual's anti-tumor immune response. Blocking CTLA-4-mediated inhibitory signaling can enhance the individual's T cell immune response to tumor cells, enhance the individual's anti-tumor activity, and produce a tumor therapeutic effect [Leach DR, et al. Science. 1996 Mar 22; 271(5256): 1734-6: Ramos-Casals M, et al. Nat Rev Dis Primers. 2020 May 7; 6(1): 38].

[0035] "Programmed cell death protein 1": Programmed cell death protein 1 (PD-1) is expressed on activated CD8+ cells. + T cells and CD4 + PD-1 is a membrane protein on the surface of T cells. Its ligand is programmed death-ligand 1 (PD-L1). PD-L1 is expressed on the surface of tumor cells and antigen-presenting cells. After PD-1 and PD-L1 bind, they are expressed on CD8 cells. + T cells and CD4 + T cells transmit inhibitory signals. These inhibitory signals limit CD8. + T cells and CD4 + T cell activation, hindering CD8 activation + T cells and CD4 + T cells kill tumor cells [Ramos-Casals M, et al. Nat Rev Dis Primers. 2020 May 7; 6(1): 38].

[0036] "Immune checkpoints": Immune checkpoints (ICs) are protein molecules that transmit inhibitory signals on T lymphocytes after recognizing their ligands. CD8 + T cells and CD4 + T cells, upon activation by dual signals, express co-inhibitory molecules, including CTLA-4 and PD1. These co-inhibitory molecules, after binding to their ligands, transmit inhibitory signals on T cells, restricting CD8. + T cells and CD4 +T cell activation. These co-inhibitory molecules are immune checkpoints, also known as immune checkpoint proteins. CTLA-4 and PD1 immune checkpoint proteins (1C proteins) [Ramos-Casals M, et al. Nat Rev Dis Primers. 2020 May 7; 6(1): 38]. ICs include, but are not limited to, CTLA-4, PD-1, PD-L1 / 2 molecules, lymphocyte-activation gene 3 (LAG-3), TIM-3 (T cell immunoglobulin and mucin domain-containing 3), TIGIT (T cell immunoreceptor with immunoglobulin and ITIM domains), and BTLA (B and T lymphocyte attenuator). Immune checkpoint blockade refers to the inhibition of ICs in the transmission of inhibitory signals on T lymphocytes. Blocking ICs enhances the individual's immune response to tumor cells, resulting in tumor therapy [Melero I, et al. Nat Rev Cancer. 2015 Aug; 15(8): 457-72].

[0037] "Immune checkpoint inhibitors": Immune checkpoint inhibitors (ICIs) are formulations that inhibit the transduction of inhibitory signals by immune checkpoint proteins on T cells. In this application, the terms "formulation" and "drug" are used interchangeably and have similar or identical meanings. ICIs may become anti-tumor drugs. They inhibit the transduction of inhibitory signals on anti-tumor T cells, thereby keeping them in a sustained and sufficient activated state to kill tumor cells, thus having a tumor therapeutic effect. CTLA-4 transmits inhibitory signals. Blocking this inhibitory signal may achieve CD8... + T cells and CD4 +T cells sustainably and efficiently kill tumor cells. CTLA-4 specific antibodies can block the transmission of CTLA-4-mediated inhibitory signals, exhibiting anti-tumor activity. CTLA-4 specific antibodies that can block CTLA-4 transmission have become an anti-tumor drug. This drug is an immune checkpoint inhibitor (ICI). ICIs are agents that target IC proteins (such as CTLA-4 and PD1) to block IC protein-mediated inhibitory signals that inhibit T cell activation. Their mechanism of action is "inhibition of inhibition to maintain activation." CTLA-4 antibodies recognize and bind to CTLA-4, blocking the interaction between CTLA-4 and CD80 and CD86 molecules. This can inhibit the transduction of T lymphocyte activation inhibitory signals, thereby keeping T cells in a sustained and fully activated state. CTLA-4 antibodies promote T cell activation by blocking the binding of CTLA-4 to CD80 or CD86 [Schaub J, Tang SC. Beyond cbeckpoint inbibitors: the three generations of immunotherapy. Clin Exp Med. 2025 Jan 21; 25(1): 43]. The bifunctional oligonucleotides (ICO-2 and ICO-3) provided in this invention are a novel type of ICI.

[0038] "Immune checkpoint antibodies": Immune checkpoint antibodies (ICI antibodies) are antibodies that target immune checkpoint proteins. After specifically binding to immune checkpoint proteins, they block the transduction of inhibitory signals in T lymphocytes. Humanized monoclonal antibodies targeting CTLA-4, PD-1, and PD-L1 are immune checkpoint antibodies. The following eight immune checkpoint antibodies have been approved by the US FDA for cancer treatment: ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cimiplimab, and dostarlimab. Ipilimumab targets CTLA-4 and is used to treat melanoma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer, and malignant pleural mesothelioma. Pembrolizumab, targeting PD-1, is used to treat 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, and Merkel cell carcinoma. Cell carcinoma, endometrial cancer, MSI-H (microsatellite highly unstable) cancer, squamous cell carcinoma of the skin, cervical cancer, squamous cell carcinoma of the head and neck, triple-negative breast cancer. Nivolumab, targeting PD-1, is used to treat melanoma, renal cell carcinoma, colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, classical Hodgkin's lymphoma, urothelial carcinoma, and esophageal cancer. Atezolizumab, targeting PD-L1, is used to treat melanoma, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, and urothelial carcinoma. Avelumab, targeting PD-L1, is indicated for Merkel cell carcinoma. Carcinoma, urothelial carcinoma, renal cell carcinoma. Durvalumab, targeting PD-L1, is used to treat non-small cell lung cancer, urothelial carcinoma, and small cell lung cancer. Cemiplimab, targeting PD-1, is used to treat squamous cell carcinoma of the skin, basal cell carcinoma, and non-small cell lung cancer.Dostarlimab, targeting PD-1, is used to treat mismatch repair deficient (dMMR) recurrent or advanced solid tumors [Schaub J, Tang SC. Clin Exp Med. 2025 Jan 21; 25(1): 43]. The bifunctional oligonucleotides (ICO-2 and ICO-3) provided in this invention may be used in combination with immune checkpoint antibodies to treat tumors.

[0039] "CTLA-4 antibody": CTLA-4 antibody is an antibody that specifically binds to CTLA4. It can block the inhibitory signal of CTLA-4 in T cell transduction, so that T lymphocytes can be fully activated by tumor antigens, which can prolong the survival of cancer patients. Ipilimumab is a fully humanized IgG1CTLA-4 monoclonal antibody [Lipson EJ., et al. Clin cancer Res. 2011 Nov 15; 17(22): 6958-62], which was approved by the US FDA in 2011 as a drug for the treatment of advanced melanoma [Sharma P. et al. Science. 2015 Apr 3; 348(6230): 56-61]. Tremelimumab is another humanized IgG2CTLA-4 monoclonal antibody [Ribas A., et al. oncologist. 2007 Jul; 12(7): 873-83]. 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 [Sho T, et al. Target Oncol. 2024 Sep; 19(5): 769-778]. CTLA-4 antibody is a CIC.

[0040] “Immune-related adverse events”: Treatment of tumors with immune checkpoint antibodies (CTLA-4 antibody, PD-1 antibody and PD-L1 antibody) can trigger a variety of diseases, which are defined as immune-related adverse events (irAEs). irAEs can affect almost any organ system. irAEs include, but are not limited to, diseases of the following organs or systems [Puzamov I, et al. J Immumother cancer. 2017 Nov 21; 5(1): 95: Ramos-Casnls M, et al. Nat Rev Dis Primers. 2020 May 7; 6(1): 38]: (1) Heart-related diseases, including myocarditis, autoimmune myocarditis, myocardial fibrosis, pericarditis, autoimmune pericarditis, pericardial effusion and pericardial tamponade. (2) Skin-related diseases, including alopecia areata / alopecia universalis, herpetic dermatitis, erythema multiforme, granuloma annulare, lichen planopilaris / planus / lichenoid dermatitis, panniculitis / erythema nodosum, pemphigoid / pemphigus, psoriasis, pyoderma gangrenosum, acute febrile neutrophilic dermatitis (Sweet syndrome), and vitiligo. (3) Endocrine-related diseases, including adrenalitis, adrenal insufficiency, cortisol deficiency, Cushing's syndrome, adrenal insufficiency, isolated ACTH deficiency, autoimmune diabetes, hyperparathyroidism, hypogonadism, hypopituitarism, autoimmune hypopituitarism, hypopituitarism, panhypopituitarism, thyroiditis, autoimmune thyroiditis, hyperthyroidism, hypothyroidism, Graves' disease, and thyrotoxicosis. (4) Gastrointestinal-related diseases, including enterocolitis, ileitis, ileocolitis, ischemic colitis, microscopic colitis, ulcerative colitis, hepatitis, autoimmune hepatitis, eosinophilic hepatitis, lymphocytic gastritis, and pancreatitis. (5) Hematologic disorders, including aplastic anemia / pure red cell aplasia, autoimmune hemolytic anemia, autoimmune neutropenia, hemophagocytic lymphohistiocytosis and immune thrombocytopenic purpura.(6) Muscle-related diseases, including myalgia, myositis, antisynthetic enzyme syndrome, bulbar myopathy, dermatomyositis, diaphragmatic lymphocytic polymyositis, polymyositis, necrotizing myopathy and orbital myositis. (7) Neurological disorders, including aseptic meningitis, encephalitis, cranial nerve involvement, bilateral hearing loss, facial paralysis, oculomotor paresis, motor neuropathy, acute generalized motor neuropathy, multifocal motor block neuropathy, myasthenia gravis, neuromyelitis optica spectrum disorders, optic neuritis, transverse myelitis, polyneuropathy, axonal sensorimotor polyneuropathy, polyneuritis, peripheral sensory neuropathy, polyradiculopathies, inflammatory demyelinating polyneuropathy, and Guillain-Barré syndrome. (8) Eye-related diseases, including conjunctivitis, episcleritis / scleritis, orbital inflammation, uveitis, anterior uveitis, choroidal retinal disease, iridocyclitis / iritis, panuveitis and posterior uveitis, and Vogt-Koyanagi-Harada syndrome. (9) Lung-related diseases, including interstitial lung disease, alveolitis, organizing pneumonia, pneumonia, pulmonary fibrosis, and pulmonary hemorrhage. (10) Kidney-related diseases, including acute tubulointerstitial nephritis, tubular acidosis, and glomerulonephritis. (11) Skeletal system-related diseases, including arthralgia / polyarthralgia, arthritis, monoarthritis, oligoarthritis, polyarthritis, enthesitis, fasciitis / eosinophilic fasciitis, Jaccoud arthropathy, polymyalgia rheumatica, psoriatic arthritis, rheumatoid arthritis, spondyloarthritis, and tenosynovitis.(12) Systemic diseases, including antiphospholipid syndrome, lupus, lupus nephropathy, subacute cutaneous lupus erythematosus, systemic lupus erythematosus, sarcoidosis, cutaneous sarcoidosis, pulmonary sarcoidosis, renal sarcoidosis, and Sjögren's syndrome. Syndrome), systemic sclerosis, vasculitis, cerebral vasculitis, cryoglobulinemia, cutaneous vasculitis, eosinophilic granulomatosis with polyangiitis, giant cell arteritis, pulmonary vasculitis, and allergic purpura. [Ramos-Casals M, et al. 2020 May 7; 6(1): 38]. Most irAEs are mild to moderate. Severe irAEs (severe colitis, pneumonia, encephalitis, toxic epidermal necrolysis, myocarditis, and type 1 diabetic ketoacidosis, etc.) are life-threatening [Puzanov I, et al. J Immunother Cancer. 2017 Nov 21; 5(1): 95]. In patients receiving CTLA-4 antibody therapy: the incidence of endocrine disorders was as high as 10%, including 4.5% of hypophysitis and 6% of thyroiditis or hypothyroidism; the incidence of heart disease was less than 1%, but could be fatal; the incidence of diarrhea was approximately 35%; the incidence of colitis was 12%; the incidence of fatal intestinal perforation due to severe colitis was as high as 1%; and the incidence of neurological disorders was 3.8% [Ramos-Casals M, et al. 2020 May 7; 6(1): 38]. The bifunctional oligonucleotides (ICO-2 and ICO-3) provided in this invention are non-antibody-targeting ICIs of CTLA-4, which largely avoid the occurrence of irAEs.

[0041] "Direct Pathogenic Effects of CTLA-4 Antibodies": CTLA-4 antibodies can cause disease by damaging normal cells through complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). Pituitary cells, particularly a subset of those secreting prolactin and thyroid-stimulating hormone, express CTLA-4. CTLA-4 antibodies recognize and bind to such cells, activating complement on their surface [Iwama S et al. Sci. Transl. Med 6, 230ra45 (2014)] or triggering ADCC. The inflammatory cascade following complement activation damages pituitary cells. ADCC also damages pituitary cells. Hypophysitis is a relatively common irAE (irAE) occurring in individuals treated with ipilimumab, with an incidence of 4.5%. The incidence of hypophysitis is 7.7% in patients receiving ipilimumab combined with PD-1 antibody therapy [Ramos-Casals M, et al. 2020 May 7; 6(1): 38]. Patients with this type of hypophysitis exhibit hypopituitarism, characterized by abnormal secretion of adrenocorticotropic hormone (ACTH) and thyroid-stimulating hormone (TSH) [Brilli L, et al. Endocrine. 2017 Dec; 58(3): 535-541]. ICO-2 and ICO-3 are non-antibody ICIs that can prevent this type of hypophysitis. The bifunctional oligonucleotides (ICO-2 and ICO-3) provided in this invention are non-antibody ICIs that target CTLA-4 and do not produce complement-dependent cytotoxicity or ADCC in normal cells.

[0042] "Anti-tumor drug": In this invention, "anti-tumor drug" can be used interchangeably with "anti-tumor agent" and has a similar or identical meaning. Anti-tumor drugs include, but are not limited to, chemotherapy drugs, tumor vaccines, tumor therapeutic antibodies, immune checkpoint antibodies, tumor angiogenesis inhibitors, oncolytic viruses, small molecule kinase inhibitors, and tumor therapeutic cells including dendritic cells, CAR-T cells, tumor-infiltrating lymphocytes, and natural killer cells. The bifunctional oligonucleotides (ICO-2 and ICO-3) provided by this invention may serve as anti-tumor drugs, therapeutically targeting tumors in individuals by enhancing their anti-tumor immune responses.

[0043] "Tumor vaccine": A tumor vaccine is a vaccine made by adding tumor antigens and tumor vaccine adjuvants [DeMaria PJ, et al. Hematol oncol Clin North Am. 2019 Apr; 33(2): 199-214]. Tumor vaccines inhibit and eliminate tumor cells by inducing an individual's anti-tumor immune response. The bifunctional oligonucleotides (ICO-2 and ICO-3) provided by this invention can be used in combination with tumor vaccines to enhance their anti-tumor immunity. Tumor vaccines include tumor antigen peptide vaccines, DNA or mRNA vaccines encoding tumor antigens, dendritic cell vaccines carrying 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 tumor vaccine are taken up and processed by antigen-presenting cells (such as dendritic cells), and then presented to the surface of antigen-presenting cells through MHC molecules. (2) T cell activation. The presented tumor antigens are recognized by T cells, activating tumor-specific T cells. B lymphocytes also participate in the activation of anti-tumor T cells. CpG ODN-activated dendritic cells and B cells have a stronger effect in activating anti-tumor T cells. (3) Tumor killing. Activated T cells migrate to the site of tumor presence, recognize and kill tumor cells expressing the corresponding antigens.

[0044] "Tumor antigen": Tumor antigen [Finn OJ. Cancer Immunol Res. 2017 May; 5(5): 347-354] can strongly stimulate tumor-specific immune responses in individuals, thereby producing a tumor therapeutic effect. Tumor antigens include tumor-associated antigens (TAAs), tumor-specific antigens [Hu Z, et al. Nat Rev Immunol. 2018 Mar; 18(3): 168-182], oncogenic viral antigens [Hu Z. et al. Nat Rev Immunol. 2018 Mar; 18(3): 168-182], 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 bifunctional oligonucleotides (ICO-2 and ICO-3) provided by this invention can enhance an individual's adaptive immune response to tumor antigens.

[0045] Neoantigens, also known as tumor neoantigens, are antigens generated by specific mutations in tumor cells. Neoantigens typically arise from gene mutations, insertions / deletions, gene rearrangements, or aberrant splicing in tumor cells. These mutations lead to changes in protein sequences, resulting in new peptides. These peptides can be presented to the cell surface by major histocompatibility complex (MHC) molecules for recognition by T cells. Unlike tumor-associated antigens (TAAs), neoantigens are expressed only in tumor cells; therefore, immune responses against neoantigens can more precisely target tumor cells while reducing damage to normal tissues. Neoantigens can be used in personalized tumor vaccines. Personalized cancer vaccines are customized based on the patient’s tumor-specific antigens and can activate a specific immune response against the tumor. They are usually designed based on the patient’s gene mutations or tumor antigens [Hu Z. et al. Nat RevImmunol 2018 Mar; 8(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].

[0046] "Tumor vaccine adjuvants" are substances that, when used in conjunction with tumor antigens, can increase the immunogenicity of tumor antigens. Tumor vaccine adjuvants can be TLR agonists, such as TLR3 agonists including polyinosinic-cytosine nucleotide (poly IC) and poly-ICLC (polyinosinic-polycytidylic acid with polylysine and carboxymethylcellulose) [Maisonneve C, et al. Proc Natl Acad Sci USA. 2014 Aug 26; 111(34): 12294-9]; TLR4 agonists including monophosphoryl lipid A (MPL) [Maisonneuve C, et al. Proc Natl Acad Sci USA. 2014 Aug 26; 111(34): 12294-9]; and TLR7 agonists including imiquimod [Maisonneuve C, et al. Proc Natl Acad Sci USA. 2014 Aug 26; 111(34): 12294-9]. 26; 111(34): 12294-91; TLR7 and TLR8 agonists including resiquimod [Maisonneuve C, et al. Proc Natl Acad Sci USA. 2014 Aug 26; 111(34): 12294-9]; TLR9 agonists including CpG ODN (CpGoligodeoxynucleotide) [Maisonneuve C, et al. Proc Natl Acad Sci USA. 2014 Aug 26; 111(34): 12294-9; Campbell JD. Methods Mol Biol. 2017; 1494: 15-27]; Cyclic dinucleotides and their analogues including c-di-GMP (cyclic di-guanosine monophosphate) [Ablasser A. Chen ZJ. Science. 2019 Mar 8;363(6431):eaat8657;Chandra D etal.STING ligand c-di-GMP improves cancer vaccination against metastatic breast cancer.Caner Immmol.Res2.901-910(2014)] and BCG, etc.The bifunctional oligonucleotides (ICO-2 and ICO-3) provided by this invention can be used as adjuvants for tumor vaccines.

[0047] "Antibodies for Tumor Therapy": The ICO-2 and ICO-3 provided by this invention can be used in combination with tumor therapeutic antibodies to treat tumors. Tumor therapeutic antibodies are antibodies that have a therapeutic effect on an individual's tumor [Scott AM et al. Nat Rev Cancer. 2012 Mar]. [22; 12(4): 278-87], these antibodies include, but are not limited to: (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) antibody targeting vascular endothelial growth factor (Bevacizumab); (5) Brentuximab (vedotin) targeting CD30; (6) Alemtuzumab (Campath) targeting CD52 and Gemtuzumabozogamicin targeting CD33; (7) antibodies targeting immune checkpoints [Schaub J, Tang SC. Clin Exp Med. 2025 Jan 21; 25(1): 43] and bispecific antibodies. The bifunctional oligonucleotides (ICO-2 and ICO-3) provided by this invention can be used in combination with antibodies for tumor treatment to treat tumors.

[0048] "Antibody targeting vascular endothelial growth factor": Bevacizumab, a monoclonal antibody targeting vascular endothelial growth factor A (VEGF-A). It is the first approved angiogenesis inhibitor. The drug was initially approved for use in combination with chemotherapy to treat metastatic colorectal cancer, and its indications have now been expanded to include a variety of malignant tumors such as metastatic breast cancer, non-small cell lung cancer, glioblastoma, renal cell carcinoma, ovarian cancer and cervical cancer [Garcia J, et al. CancerTreat Rev. 2020 Jun; 86: 102017].

[0049] “Bispecific antibodies”: Bispecific antibodies (bsAbs) are antibodies that simultaneously target two different antigens or epitopes. 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 Drug Discov. 2024 Apr; 23(4): 301-319].

[0050] Oncolytic viruses are viruses that infect and lyse tumor cells, having almost no effect on normal cells. Oncolytic viruses exert their anti-tumor effects by directly killing tumor cells and inducing an immune response [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 oncolytic herpes simplex virus type 1 (oHSV) for the treatment of recurrent melanoma. Its genome carries a gene encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) [Wang H, et al. J ImmunotherCancer. 2024 May 31; 12(5): e008025].

[0051] Small molecule kinase inhibitors: Small molecule kinase inhibitors are small molecule compounds that inhibit the activity of protein kinases. Protein kinases regulate cell differentiation, survival, and proliferation, and their overactivation contributes to tumor development, growth, and metastasis. Small molecule kinase inhibitors have significant therapeutic effects on targeted mutation-driven cancers. 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 cancers [Hussain S, et al. Eur J Pharmacol. 2024 May 5; 97e: 176484].

[0052] “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: 620-374; Gardner A, et al. Front Immunol. 2020 May 21; 11: 924], and genetically engineered chimeric antigen receptor (CAR) T cells (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], natural killer cells [Childs RW, et al. Nat Rev Drug Discov. 2015 Jul; 14(7): 487-98], and other cell preparations for cancer treatment. 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].

[0053] Synthetic lethality of cancer cells: Synthetic lethality of cancer cells refers to a strategy that uses two or more methods or drugs to kill cancer cells. Synthetic lethality is a multi-drug combination strategy. In preclinical trials, PLX4032 (a BRAF inhibitor, encorafenib) and cetuximab (an EGFR-targeting antibody) synergistically and effectively killed tumor cells. PLX4032 (a BRAF inhibitor) and gefitinib (an EGFR inhibitor) synergistically and effectively killed tumor cells [Prahallad, A. et al. Natunre. 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 as a synthetic lethality therapy for the treatment of BRAF-mutant metastatic colorectal cancer. Synthetic lethality strategies for cancer cells include dual synthetic lethality, multi-synthetic lethality, and compound synthetic lethality [Ryan CJ, et al. NatGenet. 2023 Dec; 55(12): 2039-2048]. The bifunctional oligonucleotides (ICO-2 and ICO-3) provided in this invention can be used in combination with antitumor agents (drugs) to treat tumors and promote the synthetic lethality of cancer cells. In October 2022, the US 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 lethality treatment method for cancer cells [Sho T, et al. Target Oncol. 2024 Sep; 19(5): 769-778].

[0054] "Local tumor administration": Applying antitumor agents (drugs) locally to the tumor may improve their antitumor efficacy and avoid side effects (such as irAEs) [Fransen MF, et al. Clin. Cancer Res 19, 5381-5389 (2013); Marabelle A, et al. Clin. Cancer Res 19, 5261-5263 (2013)]. The route of administration may affect its toxicity. 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-Baarfi I, et al. Sci Immunol. 2022 May 27; 7(71): eabn5859]. Administration within the tumor and in the tumor lymph node drainage area are both local tumor administration strategies. The bifunctional oligonucleotides (ICO-2 and ICO-3) provided by this invention can be applied via intratumoral and tumor lymph node drainage areas, as well as via mucosa, peritoneum, intrathecal, intramuscular, subcutaneous, intradermal, local, peritumoral, and intra-lymph node routes.

[0055] "Delivery System": The bifunctional oligonucleotides (ICO-2 and ICO-3) provided by this invention can be delivered using a delivery system. Delivery systems for ICO-2 and ICO-3 include, but are not limited to, lipid nanoparticles (LNPs) and lipoplexes [Cnllis PR, Felgner PL. The 60-year evolution of lipid nanoparticles for nucleic acid delivery. Nat Rev Drug Diseov. 2024 Sep; 23(9): 709-722]. Attached image description:

[0056] Figure 1. Identification of bifunctional oligonucleotides.

[0057] Three bifunctional oligonucleotides (ICO-1, 23 bp; ICO-2, 25 bp; ICO-3, 25 bp) were identified by 2% agarose gel electrophoresis. DM, DNA bar (Dongsheng Biotechnology, China), molecular weights were 2000, 1000, 750, 500, 250, and 100 bp, respectively.

[0058] Figure 2. Activation effect of bifunctional oligonucleotide (ICO-2) on B cells.

[0059] Experimental Procedure. Splenocytes from unsensitized BALB / c mice were co-cultured with ICO-2, ICO-1, or Med (1640 medium). After 24 hours, they were co-stained with CD19-PE monoclonal antibody, CD40-FITC monoclonal antibody, and CD86-APC monoclonal antibody. Flow cytometry analysis was performed. Gating Strategy: P1 was the viable cell gate; CD19 cells were delineated from the P1 gate. + Cellular phylum (R1), CD19 is further circled from R1 phylum. hi Cellular phylum (R2). (C) CD19 expression. Analysis of CD19 in the R1 phylum. + Percentage of cells and CD19 + The expression level of CD19 (mean fluorescence intensity, MFI) was analyzed using the R2 gate. hi+ Cell percentage (%) and CD19 expression level (MFI). CD86 expression. CD86 analysis. + CD19 + Percentage of cells (%) and CD86 in CD19 + Cellular expression levels (MFI). CD40 expression. Analysis of CD40. + CD19 + Percentage of cells (%) and CD40 in CD19 + Expression level on cells (MFI).

[0060] Figure 3. Activation effect of bifunctional oligonucleotide (ICO-3) on B cells.

[0061] Experimental Procedure. Splenocytes from unsensitized BALB / c mice were co-cultured with ICO-3, ICO-1, or Med (1640 medium). After 24 hours, they were co-stained with CD19-PE monoclonal antibody, CD40-FITC monoclonal antibody, and CD86-APC monoclonal antibody. Flow cytometry analysis was performed. Gating Strategy: P1 was the viable cell gate; CD19 cells were delineated from the P1 gate. + Cellular phylum (R1), CD19 is further circled from R1 phylum. hi Cellular phylum (R2). (C) CD19 expression. Analysis of the percentage of CD19+ cells and CD19 expression in the R1 phylum. + The expression level of CD19 (mean fluorescence intensity, MFI) was analyzed using the R2 gate. hi+ Cell percentage (%) and CD19 expression level (MFI). CD86 expression. CD86 analysis. + CD19 + Percentage of cells (%) and CD86 in CD19 +Cellular expression levels (MFI). CD40 expression. Analysis of CD40. + CD19 + Percentage of cells (%) and CD40 in CD19 + Expression level on cells (MFI).

[0062] Figure 4. Activation effect of bifunctional oligonucleotide (ICO-2) on dendritic cells.

[0063] Experimental Procedure. BALB / c mice were subcutaneously injected with ICO-2, ICO-1, or PBS into the medial right thigh near the inguinal lymph node (DLN). 24 hours later, the mice were sacrificed, and inguinal lymph node cells were isolated. Flow cytometry was used to detect cells co-labeled with CD11c-FITC, CD80-PE, and CD86-APC monoclonal antibodies. CD11c + Cell count. Circle CD11c from the phylum P1 (living cell type). + Cellular phylum (R1), analysis of CD11c + Cell percentage (%) and number (counts). Expression of CD80 and CD86. Analysis of CD80 from the R1 gate. + CD11c + Cells, CD86 + CD11c + Percentage of cells and expression levels of CD80 and CD86 on CD11c+ cells (MFI).

[0064] Figure 5. Activation effect of bifunctional oligonucleotide (ICO-3) on dendritic cells.

[0065] Experimental Procedure. BALB / c mice were subcutaneously injected with ICO-3, ICO-1, or PBS into the medial right thigh near the inguinal lymph node (DLN). 24 hours later, the mice were sacrificed, and inguinal lymph node cells were isolated. Cells co-labeled with CD11c-FITC, CD80-PE, and CD86-APC monoclonal antibodies were detected by flow cytometry. (B) CD11c + Cell count. Circle CD11c from the phylum P1 (living cell type). + Cellular phylum (R1), analysis of CD11c + Cell percentage (%) and number (counts). (C) Expression of CD80 and CD86. Analysis of CD80 from the R1 gate. + CD11c + Cells, CD86 + CD11c +The percentage of cells and CD80, CD86 in CD11c + Expression level on cells (MFI).

[0066] Figure 6. The inhibitory effect of bifunctional oligonucleotide (ICO-2) on CTLA-4 expression in T cells.

[0067] Experimental Procedure. Unsensitized BALB / c mice were subcutaneously injected with an inactivated influenza virus vaccine (Flu vaccine) into the medial right thigh near the inguinal lymph node drainage area. 48 hours later, ICO-2, ICO-1, or PBS was injected at the injection site. 72 hours later, the mice were sacrificed, and inguinal lymph node (ILN) cells were isolated. The number of CD4+ T cells co-labeled with CD4-FITC and CD152-PE monoclonal antibodies was detected by flow cytometry. CD4+ T cells were circled from the live cell phylum (P1). + Cellular phylum (R1). Analysis of CD4 in the R1 phylum. + Percentage of cells in ILNs (%). CTLA-4 expression. CTLA-4 analysis from the R1 gate. + CD4 + Cell percentage (%) and CD4 + CTLA-4 expression level on cells (MFI).

[0068] Figure 7. Inhibitory effect of bifunctional oligonucleotide (ICO-3) on T cell expression of CTLA-4.

[0069] Experimental Procedure. Unsensitized BALB / c mice were subcutaneously injected with an inactivated influenza virus vaccine (Flu vaccine) into the medial right thigh near the inguinal lymph node drainage area. 48 hours later, ICO-3, ICO-1, or PBS was injected at the injection site. 72 hours later, the mice were sacrificed, and inguinal lymph node (ILN) cells were isolated. Flow cytometry was used to detect the number of CD4+ T cells co-labeled with CD4-FITC and CD152-PE monoclonal antibodies. CD4+ T cells were circled from the live cell phylum (P1). + Cellular phylum (R1). Analysis of CD4 in the R1 phylum. + Percentage of cells in ILNs (%). CTLA-4 expression. CTLA-4 analysis from the R1 gate. + CID4 + Cell percentage (%) and CD4 + CTLA-4 expression level on cells (MFI).

[0070] Figure 8. The inhibitory effect of bifunctional oligonucleotide (ICO-2) on tumor growth and CTLA-4 expression on T cells in bilateral tumor-bearing mice.

[0071] (A) Experimental Procedure. B16 cells (5 × 10⁶ cells) were subcutaneously inoculated into the medial thighs of C57BL / 6 mice (n = 5 per group) near the inguinal lymphatic drainage area. 5 (A) Inoculation: On days 1, 3, 5, 7, and 9 post-inoculation, ICO-2 was injected into the right inoculation site, and PBS was injected into the left. (B) Lymph node size and lymphocyte count: On day 10 post-inoculation, bilateral inguinal lymph nodes (ILNs) were collected from mice, and ILN cell suspensions were prepared and counted. (C) Tumor size and weight: On day 10 post-inoculation, tumors were collected, photographed, and weighed. (D) CTLA-4 expression: Flow cytometry was used to detect ILN cells co-labeled with CD4-FITC, CD8-APC, and CD152-PE monoclonal antibodies. CTLA-4 expression in P1 (live cell phylum) was analyzed. + Percentage (%) of ILN cells and their CTLA-4 expression level (MFI). Circle CD4 from P1. + Cellular phylum (R1) and CD8 + Cellular phylum (R2). Analysis of CTLA-4 from the R1 phylum. + CD4 + Cell percentage (%) and its CTLA-4 expression level (MFI). CTLA-4 was analyzed from the R2 gate. + CD8 + The percentage of cells (%) and their CTLA-4 expression level (MFI).

[0072] Figure 9. Inhibitory effect of bifunctional oligonucleotide (ICO-2) on tumor growth in unilateral melanoma-bearing mice.

[0073] Experimental Procedure. On Day 0, B16 cells (1×10⁶) were subcutaneously inoculated into the medial right thigh of C57BL / 6 mice near the groin lymphatic drainage area. 5 On days 1, 3, 5, 7, and 9, ICO-2 (20 μg / mouse) or PBS was injected at the inoculation site. Tumor volume and survival rate in mice were measured every other day starting on day 19 post-inoculation (when tumors were measurable). Each broken line represents the dynamic volume of a mouse's tumor. Mouse mortality was recorded starting on day 32 post-inoculation (when mice began to die). Tumor formation in mice re-inoculated with B16 cells was assessed. On day 100, two mice that had not developed tumors after ICO-2 injection were re-inoculated (re-challenge) with B16 cells (1 × 10⁻⁶). 5 ). Initial seeding of B16 cells (1×10⁻⁶) 5C57BL / 6 mice (n=9) were used as a control. Neither group (Re-challenge nor Challenge) received ICO-2 injections. Tumor growth and death were recorded.

[0074] Figure 10. Inhibitory effect of bifunctional oligonucleotide (ICO-2) on tumor growth in mice bearing colon cancer cells.

[0075] (A) Experimental Procedure. CT26 cells (5 × 10⁻⁶) were subcutaneously injected into the medial right thigh near the groin lymphatic drainage area of ​​BALB / c mice (n = 10 / group). 5 / mouse). ICO-2 (20 μg / mouse) or PBS was injected into the injection site on days 1, 3, 5, 7, and 9 post-inoculation. Tumor size was measured every other day starting on day 13 post-inoculation (when the tumor was measurable). Mice mortality was recorded starting on day 32 post-inoculation (when mice began to die). (B) Tumor volume and survival rate of mice. Tumor volume (mm) 3 = Length (longest tumor diameter in mm) × Width (shortest tumor diameter in mm) 2 × 0.52.

[0076] Figure 11. Inhibitory effect of bifunctional oligonucleotide (ICO-2) on tumor growth formed by lung cancer cells.

[0077] On day 0, Lewis lung cancer cells (LLCC) (1×10⁻⁶) were subcutaneously inoculated into the medial right thigh near the groin lymph node drainage area of ​​C57BL / 6 mice (n=5). 5 On days 1, 3, 5, 7, and 9, ICO-2 (20 μg / animal) or PBS was injected at the inoculation site. Tumor size was measured starting on day 13 post-inoculation (when the tumor could be measured). Tumor volume (mm²) 3 = Length (diameter of the longest tumor in mm) × Width (diameter of the shortest tumor in mm) 2 ×0.52.

[0078] Figure 12. Inhibitory effect of bifunctional oligonucleotide (ICO-3) on the growth of melanoma formed by B16 cells.

[0079] On day 0, B16 cells (1×10⁻⁶) were subcutaneously inoculated into the medial right thigh of C57BL / 6 mice near the groin lymphatic drainage area. 5 On days 1, 3, 5, 7, and 9, ICO-3 (20 μg / animal) or PBS was injected at the inoculation site. Tumor size was measured every other day starting on day 13 post-inoculation. Tumor growth was recorded. Tumor volume (mm) 3 = Length (longest tumor diameter in mm) × Width (shortest tumor diameter in mm) 2 ×0.52.

[0080] Figure 13. Inhibitory effect of bifunctional oligonucleotide (ICO-3) on tumor growth in mice bearing colon cancer cells.

[0081] CT26 cells (5 × 10⁻⁶) were subcutaneously injected into the right medial thigh near the inguinal lymph node drainage area of ​​BALB / c mice (n = 8 / group). 5 / mouse). On days 1, 3, 5, 7, and 9 post-inoculation, mice were injected with ICO-3 (20 μg / mouse) or PBS at the injection site. Tumor size was measured every other day starting on day 13 post-inoculation. Tumor volume (mm) 3 = Length (longest tumor diameter in mm) × Width (shortest tumor diameter in mm) 2 ×0.52.

[0082] Figure 14. Inhibitory effect of bifunctional oligonucleotide (ICO-3) on tumor growth formed by lung cancer cells.

[0083] On day 0, Lewis lung cancer cells (LLCC) (1×10⁻⁶) were subcutaneously inoculated into the medial right thigh near the groin lymph node drainage area of ​​C57BL / 6 mice (n=8). 5 On days 1, 3, 5, 7, and 9, ICO-3 (20 μg / animal) or PBS was injected at the inoculation site. Tumor size was measured starting on day 13 post-inoculation. Tumor volume (mm²) 3 = Length (diameter of the longest tumor in mm) × Width (diameter of the shortest tumor in mm) 2 ×0.52. Specific implementation methods:

[0084] Example 1: Synthesis and Detection of Oligonucleotides (ICO-2, ICO-3)

[0085] Having a sequence list <400> 1 (SEQ ID NO: 1) is a single-stranded deoxy oligonucleotide (ICO-2) with the sequence shown (5'-TTCTACCTCGTGTATTCGTGTCCTG-3'), having the sequence listed in the sequence table. <400> The single-stranded deoxyribonucleotide (ICO-3) with the sequence (5'-TGACCTCGTGTTCTACCTCGTGTAT-3') and the single-stranded deoxyribonucleotide (ICO-1) with the sequence (5'-GAGCGGTGTTCAGGTCGTCAGGA-3') were synthesized by Takara Biotech (Takara Biotech, Dalian, China), with a fully thiolated backbone. ICO-2, ICO-3, and ICO-1 were dissolved in sterile PBS (8.0 g / L sodium chloride, 0.2 g / L potassium chloride, 1.44 g / L disodium hydrogen phosphate, and 0.24 g / L sodium dihydrogen phosphate, pH = 7.4). Endotoxin levels were <5 EU / mg as determined by the horseshoe crab cell lysis method. Quantification was performed using a spectrophotometer at 260 nm. The integrity and content of ICO-2, ICO-3, and ICO-1 were assessed by 2% agarose gel electrophoresis. Repackage and store at -20℃.

[0086] Example 2: Activation of B cells by bifunctional oligonucleotide (ICO-2)

[0087] 2.1. Materials

[0088] 2.1.1 Mice

[0089] Female BALB / c mice (6-8 weeks old) were purchased from Yisi Laboratory Animal Technology Co., Ltd., Changchun, China. All mice were housed in a specific pathogen-free (SPF) environment at the Experimental Animal Center of Jilin University, with free access to food and water. All animal experimental procedures involved in this study strictly followed the National Research Council's Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Ethics Committee of the School of Basic Medical Sciences, Jilin University.

[0090] 2.1.2. Oligonucleotides

[0091] ICO-2 and ICO-1 as described in Example 1.

[0092] 2.1.3. Cell Culture Medium

[0093] Complete RPMI medium (Gibco) containing 10% (v / v) fetal bovine serum (FBS), 100 IU penicillin / ml and 100 IU streptomycin / ml. Serum-free RPMI medium, without fetal bovine serum, containing 100 IU penicillin / ml and 100 IU streptomycin / ml. RPMI medium with 2% FBS, containing 2% (v / v) FBS, 100 IU penicillin / ml and 100 IU streptomycin / ml.

[0094] 2.2. Methods

[0095] 6-8 week old BALB / c mice were euthanized. They were immersed in 75% ethanol for 2 minutes. The spleen was removed using ophthalmic scissors and forceps. It was placed in a petri dish (on ice) containing 6 ml of pre-chilled complete RPMI medium. The spleen was ground using two ground glass slides. The ground spleen cells were resuspended in complete RPMI medium. The suspension was filtered through a 300-mesh filter into a 10 ml centrifuge tube. The tube was centrifuged (using a TDL-5000bR centrifuge) for 5 minutes at 900 rpm. The supernatant was discarded after centrifugation. 3 ml of pre-chilled ACK solution (0.155 mol / L ammonium chloride, 0.01 mol / L potassium bicarbonate, 0.1 mol / L EDTA, pH 7.4) was added, and the mixture was thoroughly mixed. After 2 minutes, complete RPMI medium was added to a final volume of 10 ml. The tube was centrifuged (1020 rpm, 5 minutes), and the supernatant was discarded. The cells were resuspended in 1 ml of complete RPMI medium. The mixture was then mixed. Cells were counted. 1 ml of spleen cells (3 × 10) 6Add the cell suspension (3 μg / ml) to a 24-well plate (NEST, China), then add 100 μl of ICO-2 (3 μg / ml) or ICO-1 (3 μg / ml), respectively. Incubate at 37°C in a 5% CO2 cell culture incubator for 24 h. Collect the cell suspension using a 1.5 ml Eppendorf tube. Centrifuge for 5 min at 3000 rpm. Discard the supernatant. Resuspend the cells in 50 μl of PBS (biopico, China) containing 2% FBS. Add CD19-PE (Rat anti-Mouse, Cat#557399, BD Biosciences, NJ, USA), CD40-FITC (Rat anti-Mouse, Cat#553790, BD Biosciences, NJ, USA), or CD86-APC (Rat anti-Mouse, Cat#558703, BD Biosciences, NJ, USA) diluted 1:10 with PBS, and incubate on ice for 30 min. Wash with PBS and centrifuge at 3000 rpm for 5 min. Resuspend the cells in 200 μl of PBS containing 2% FBS. Analyze 50,000 viable cells using a flow cytometer (BD, Accuri C6).

[0096] 2.3 Results

[0097] The results (Figure 1.2) show that, as in the sequence list... <400> The bifunctional oligonucleotide (ICO-2) shown in Figure 1 stimulates upregulation of CD19 expression in spleen cells. Upon stimulation by ICO-2, CD19 expression in spleen cells... + Two CD19 cells (R1 phylum) were observed, one smaller and one larger than normal. + Cell population analysis suggests that ICO-2 can induce blastogenesis in B lymphocytes. ICO-2 can upregulate CD19. + Cells express CD19, making them highly CD19-expressing cells (CD19 hi ICO-2 can increase CD19. + The number of CD19hi cells in cells. CD19 is a regulated B cell-specific surface protein. Upregulation of CD19 can activate B cells. ICO-2 can significantly upregulate CD86 expression in CD19hi cells and also increase CD40 expression. + CD19 + Percentage of cells. CD86 and CD40 are both co-stimulatory molecules on the surface of B cells, and their upregulation can indicate B cell activation. These results indicate that ICO-2 can activate B cells, enabling them to more effectively cooperate with anti-tumor T cells to kill tumor cells, thus exhibiting a tumor therapeutic effect.

[0098] Example 3: Activation of B cells by bifunctional oligonucleotides (ICO-3)

[0099] 3.1. Materials

[0100] 3.1.1 Mice

[0101] Female BALB / c mice (6-8 weeks old) were purchased from Yisi Laboratory Animal Technology Co., Ltd., Changchun, China. All mice were housed in an SPF (specific pathogen-free) environment at the Jilin University Laboratory Animal Center, with free access to food and water. All animal experimental procedures involved in this study strictly followed the National Research Council's Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Ethics Committee of the School of Basic Medical Sciences, Jilin University.

[0102] 3.1.2. Oligonucleotides

[0103] ICO-3 and ICO-1 as described in Example 1.

[0104] 3.1.3. Cell Culture Medium

[0105] Complete RPMI medium (Gibco) containing 10% (v / v) fetal bovine serum (FCS), 100 IU penicillin / ml and 100 IU streptomycin / ml. Serum-free RPMI medium, without fetal bovine serum, containing 100 IU penicillin / ml and 100 IU streptomycin / ml. RPMI medium with 2% FBS, containing 2% (v / v) FBS, 100 IU penicillin / ml and 100 IU streptomycin / ml.

[0106] 3.2 Methods

[0107] 6-8 week old BALB / c mice were euthanized. They were immersed in 75% ethanol for 2 minutes. The spleen was removed using ophthalmic scissors and forceps. It was placed in a petri dish (on ice) containing 6 ml of pre-chilled complete RPMI medium. The spleen was ground using two ground glass slides. The ground spleen cells were resuspended in complete RPMI medium. The suspension was filtered through a 300-mesh filter into a 10 ml centrifuge tube. The tube was centrifuged (900 rpm) for 5 minutes using a TDL-5000bR centrifuge. The supernatant was discarded after centrifugation. 3 ml of pre-chilled ACK solution (0.155 mol / L ammonium chloride, 0.01 mol / L potassium bicarbonate, 0.1 mol / L EDTA, pH 7.4) was added, and the mixture was thoroughly mixed. After 2 minutes, complete RPMI medium was added to a final volume of 10 ml. The tube was centrifuged (1020 rpm, 5 minutes), and the supernatant was discarded. The cells were resuspended in 1 ml of complete RPMI medium. The mixture was then mixed. Cells were counted. 1 ml of spleen cells (3 × 10) 6 Add the cell suspension (3 μg / ml) to a 24-well plate (NEST, China), then add 100 μl of ICO-3 or ICO-1 (3 μg / ml). Incubate at 37°C in a 5% CO2 cell culture incubator for 24 h. Collect the cell suspension using a 1.5 ml Eppendorf tube. Centrifuge for 5 minutes at 3000 rpm. Discard the supernatant. Resuspend the cells in 50 μl of PBS containing 2% FBS (Biopico, China). Add CD19-PE (Rat anti-Mouse, Cat#557399, BD Biosciences, NJ, USA), CD40-FITC (Rat anti-Mouse, Cat#553790, BD Biosciences, NJ, USA), or CD86-APC (Rat anti-Mouse, Cat#558703, BD Biosciences, NJ, USA) diluted 1:10 with PBS, and incubate on ice for 30 min. Wash with PBS and centrifuge at 3000 rpm for 5 min. Resuspend the cells in 200 μl of PBS containing 2% FBS. Analyze 50,000 viable cells using a flow cytometer (BD, Accuri C6).

[0108] 3.3 Results

[0109] The results (Figure 1.3) show that, as in the sequence list <400> The bifunctional oligonucleotide (ICO-3) shown in Figure 2 stimulated upregulation of CD19 expression in splenocytes. ICO-3 both increased the number of CD19+ cells in splenocytes and upregulated CD19 expression. Upon stimulation with ICO-3, two CD19+ cells (R1 phylum) in the splenocytes showed two different sizes: one smaller and one larger. +Cell population analysis suggests that ICO-3 can induce blastogenesis in B lymphocytes. ICO-3 can upregulate CD19 expression in CD19+ cells, making them highly CD19-expressing cells. hi ICO-3 can increase CD19. + CD19 in cells hi Cell count. CD19 is a regulated B cell-specific surface protein. Upregulation of CD19 can activate B cells. ICO-3 can significantly increase CD86. + CD19 + Percentage of cells. ICO-3 can increase CD40. + CD19 + Percentage of cells. CD86 and CD40 are both co-stimulatory molecules on the surface of B cells, and their upregulation can indicate B cell activation. These results indicate that ICO-3 can activate B cells, enabling them to more effectively cooperate with anti-tumor T cells to kill tumor cells, thus exhibiting a tumor therapeutic effect.

[0110] Example 4: Activation of dendritic cells by bifunctional oligonucleotide (ICO-2)

[0111] 4.1. Materials

[0112] 4.1.1 Mice

[0113] Female BALB / c mice (6-8 weeks old) were purchased from Yisi Laboratory Animal Technology Co., Ltd., Changchun, China. All mice were housed in an SPF (specific pathogen free) environment at the Experimental Animal Center of Jilin University, with free access to food and water. All animal experimental procedures involved in this study strictly followed the guidelines of the National Research Council's Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Ethics Committee of the School of Basic Medical Sciences, Jilin University.

[0114] 4.1.2. ICO-2 and ICO-1 as described in Example 1.

[0115] 4.1.3. Cell Culture Medium

[0116] Complete RPMI medium (Gibco) containing 10% (v / v) fetal bovine serum (FCS), 100 IU penicillin / ml and 100 IU streptomycin / ml. Serum-free RPMI medium, without fetal bovine serum, containing 100 IU penicillin / ml and 100 IU streptomycin / ml. RPMI medium with 2% FBS, containing 2% (v / v) FBS, 100 IU penicillin / ml and 100 IU streptomycin / ml.

[0117] 4.2 Methods

[0118] In BALB / c mice, 100 μl of PBS containing either ICO-2 (10 μg / mouse) or ICO-1 (10 μg / mouse) was subcutaneously injected into the medial thigh of the right thigh (near the inguinal lymph node drainage area). After 24 h, inguinal lymph nodes were harvested. The cells were homogenized into a single-cell suspension using a ground glass slide in an ice-cold Petri dish containing 1 ml of 10% FBS RPMI 1640. The cell suspension was filtered through a 300-mesh filter into a 1.5 ml Eppendorf tube. The cells were centrifuged at 3000 rpm for 5 minutes. The supernatant was discarded after centrifugation. The cells were resuspended in 50 μl of PBS (Biopico, China) containing 2% FBS. Add CD11c-FITC (Hamster anti-Mouse, Cat#553801, BD Biosciences, NJ, USA), CD80-PE (Hamster anti-Mouse, Cat#104708, Biolegend, CA, USA), and CD86-APC (Rat anti-Mouse, Cat#558703, BD Biosciences, NJ, USA) diluted 1:10 with PBS and incubate on ice for 30 min. Wash with PBS and centrifuge at 3000 rpm for 5 min. Resuspend the cells in 200 μl of PBS containing 2% FBS. Analyze 50,000 viable cells using a flow cytometer (Accuri C6Plus, BD Biosciences, NJ, USA).

[0119] 4.3 Results

[0120] result( Figure 4 The display shows that it has a sequence list. <400> The bifunctional oligonucleotide (ICO-2) with the sequence shown in Figure 1 can increase CD80 in lymph nodes. + CD11c + The number of cells can be increased, and CD80 can be upregulated in CD11c. + ICO-2 expression on cells. Furthermore, ICO-2 can increase CD86 expression. + CD11c + The number of cells was increased, and CD86 was upregulated in CD11c. +Cellular expression. CD80 and CD86 are co-stimulatory molecules expressed on the surface of dendritic cells (DCs), and their upregulation marks the activation of DCs. DCs with high expression of co-stimulatory molecules can more effectively provide the second signal for T cell activation. These results suggest that ICO-2 can activate DCs, transforming them into antigen-presenting cells that are more efficient at activating T cells.

[0121] Example 5: Activation of dendritic cells by bifunctional oligonucleotides (ICO-3)

[0122] 5.1. Materials

[0123] 5.1.1 Mice

[0124] Female BALB / c mice (6-8 weeks old) were purchased from Yisi Laboratory Animal Technology Co., Ltd., Changchun, China. All mice were housed in an SPF (specific pathogen free) environment at the Experimental Animal Center of Jilin University, with free access to food and water. All animal experimental procedures involved in this study strictly followed the National Research Council's Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Ethics Committee of the School of Basic Medical Sciences, Jilin University.

[0125] 5.1.2. Oligonucleotides

[0126] As described in Example 1, ICO-3 and ICO-1

[0127] 5.1.3. Cell Culture Medium

[0128] Complete RPMI medium (Gibco) containing 10% (v / v) fetal bovine serum (FCS), 100 IU penicillin / ml and 100 IU streptomycin / ml. Serum-free RPMI medium, without fetal bovine serum, containing 100 IU penicillin / ml and 100 IU streptomycin / ml. RPMI medium with 2% FBS, containing 2% (v / v) FBS, 100 IU penicillin / ml and 100 IU streptomycin / ml.

[0129] 5.2 Methods

[0130] In BALB / c mice, 100 μl of PBS containing either ICO-3 (10 μg / mouse) or ICO-1 (10 μg / mouse) was subcutaneously injected into the medial thigh of the right thigh (near the inguinal lymph node drainage area). After 24 h, inguinal lymph nodes were harvested. The cells were homogenized into a single-cell suspension using a ground glass slide in an ice-cold petri dish containing 1 ml of 10% FBS RPMI 1640. The cell suspension was filtered through a 300-mesh filter into a 1.5 ml Eppendorf tube. The cells were centrifuged at 3000 rpm for 5 minutes. The supernatant was discarded after centrifugation. The cells were resuspended in 50 μl of PBS (Biopico, China) containing 2% FBS. Add CD11c-FITC (Hamster anti-Mouse, Cat#553801, BD Biosciences, NJ, USA), CD80-PE (Hamster anti-Mouse, Cat#104708, Biolegend, CA, USA), and CD86-APC (Rat anti-Mouse, Cat#558703, BD Biosciences, NJ, USA) diluted 1:10 with PBS, and incubate on ice for 30 min. Wash with PBS and centrifuge at 3000 rpm for 5 min. Resuspend the cells in 200 μl of PBS containing 2% FBS. Analyze 50,000 viable cells using a flow cytometer (Accuri C6 Plus, BD Biosciences, NJ, USA).

[0131] 5.3 Results

[0132] result( Figure 5 The display shows that it has a sequence list. <400> The bifunctional oligonucleotide (ICO-3) with the sequence shown in Figure 2 can increase CD80 in lymph nodes. + CD11c + The number of cells can be increased, and CD80 can be upregulated in CD11c. + Cellular expression. CD80 is a co-stimulatory molecule expressed on the surface of dendritic cells (DCs), and its upregulation marks DC activation. DCs with high expression of the co-stimulatory molecule can more effectively provide the second signal for T cell activation. The above results demonstrate that ICO-3 can activate DCs, transforming them into antigen-presenting cells that are more efficient at activating T cells.

[0133] Example 6: Inhibitory effect of bifunctional oligonucleotide (ICO-2) on CTLA-4 expression in T cells

[0134] 6.1. Materials

[0135] 6.1.1 Mice

[0136] Female BALB / c mice (6-8 weeks old) were purchased from Yisi Laboratory Animal Technology Co., Ltd., Changchun, China. All mice were housed in an SPF (specific pathogen free) environment at the Experimental Animal Center of Jilin University, with free access to food and water. All animal experimental procedures involved in this study strictly followed the National Research Council's Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Ethics Committee of the School of Basic Medical Sciences, Jilin University.

[0137] 6.1.2 Influenza vaccine

[0138] FM1 influenza virus vaccine (Flu Vaccine). The antigen is inactivated FM1 influenza virus with a hemagglutination titer of 1024 HAU / 200 μl. The adjuvant is T9 oil-in-water emulsion. Prepared by the Department of Immunology, School of Basic Medical Sciences, Jilin University.

[0139] 6.1.3. Oligonucleotides

[0140] As described in Example 1, ICO-2 and ICO-1

[0141] 6.1.4. Cell Culture Medium

[0142] Complete RPMI medium (Gibco) containing 10% (v / v) fetal bovine serum, 100 IU penicillin / ml and 100 IU streptomycin / ml. Serum-free RPMI medium, without fetal bovine serum, containing 100 IU penicillin / ml and 100 IU streptomycin / ml. RPMI medium with 2% FBS, containing 2% (v / v) FBS, 100 IU penicillin / ml and 100 IU streptomycin / ml.

[0143] 6.2 Methods

[0144] BALB / c mice were subcutaneously inoculated with 100 μl of our laboratory-prepared inactivated FM1 influenza virus vaccine (Flu Vaccine) on the medial right thigh (near the inguinal lymph node drainage area). The antigen of Flu Vaccine was inactivated FM1 influenza virus with a hemagglutination titer of 1024 HAU / 200 μl. The adjuvant was T9 oil-in-water emulsion. 48 h later, 100 μl of PBS containing ICO-2 and ICO-1 (10 μg / mouse) was injected at the same site. 24 h later, lymph node cells were isolated from the ipsilateral inguinal lymph node and ground into a single-cell suspension using a ground glass slide in a pre-chilled petri dish containing 1 ml of 10% FBBS-RPMI 1640. The cell suspension was filtered through a 300-mesh filter into a 1.5 ml Eppendorf tube. Centrifuged for 5 minutes at 3000 rpm. The supernatant was discarded after centrifugation. Cells were resuspended in 50 μl of PBS (Biopico, China) containing 2% FBS. In the lymph node cell suspension, CD4-FITC (Ratanti-Mouse, Cat#553046, BD Biosciences, NJ, USA) and CD152-PE (Hamster anti-Mouse, Cat#553720, BD Biosciences, NJ, USA) diluted 1:10 with PBS were added, and the mixture was incubated on ice for 30 min. Cells were washed with PBS and centrifuged at 3000 rpm for 5 min. Cells were resuspended in 200 μl of PBS containing 2% FBS. 50,000 viable cells were analyzed using flow cytometry (Accuri C6Plus, BD Biosciences, NJ, USA).

[0145] 6.3. Results

[0146] result( Figure 6 The display shows that it has a sequence list. <400> The bifunctional oligonucleotide (ICO-2) of the sequence shown in Figure 1 can downregulate CTLA-4 in CD4+. + This indicates that ICO-2 can inhibit CD4 expression on T cells. + T cells are in a state of continuous activation. CD4 cells are in this state. + T cells can assist tumor-specific CD8 cells. + T cells kill tumor cells.

[0147] Example 7: Inhibitory effect of bifunctional oligonucleotide (ICO-3) on CTLA-4 expression in T cells

[0148] 7.1. Materials

[0149] 7.1.1 Mice

[0150] Female BALB / c mice (6-8 weeks old) were purchased from Yisi Laboratory Animal Technology Co., Ltd., Changchun, China. All mice were housed in an SPF (specific pathogen free) environment at the Experimental Animal Center of Jilin University, with free access to food and water. All animal experimental procedures involved in this study strictly followed the National Research Council's Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Ethics Committee of the School of Basic Medical Sciences, Jilin University.

[0151] 7.1.2 Influenza vaccine

[0152] FM1 influenza virus vaccine (Flu Vaccine). The antigen is inactivated FM1 influenza virus with a hemagglutination titer of 1024 HAU / 200 μl. The adjuvant is T9 oil-in-water emulsion. Prepared by the Department of Immunology, School of Basic Medical Sciences, Jilin University.

[0153] 7.1.3. Oligonucleotides

[0154] ICO-3 and ICO-1 as described in Example 1.

[0155] 7.1.4. Cell Culture Medium

[0156] Complete RPMI medium (Gibco) containing 10% (v / v) fetal bovine serum (FCS), 100 IU penicillin / ml and 100 IU streptomycin / ml. Serum-free RPMI medium, without fetal bovine serum, containing 100 IU penicillin / ml and 100 IU streptomycin / ml. RPMI medium with 2% FBS, containing 2% (v / v) FBS, 100 IU penicillin / ml and 100 IU streptomycin / ml.

[0157] 7.2 Methods

[0158] BALB / c mice were subcutaneously inoculated with 100 μl of our laboratory-prepared inactivated FM1 influenza virus vaccine (Flu Vaccine) on the medial right thigh (near the inguinal lymph node drainage area). The antigen of Flu Vaccine was inactivated FM1 influenza virus with a hemagglutination titer of 1024 HAU / 200 μl. The adjuvant was T9 oil-in-water emulsion. 48 h later, 100 μl of PBS containing ICO-3 (10 μg / mouse) and ICO-1 (10 μg / mouse) was injected at the same site. 24 h later, lymph node cells were isolated from the ipsilateral inguinal lymph node and ground into a single-cell suspension using a ground glass slide in a petri dish pre-chilled on ice with 1 ml of 10% FBS RPMI 1640. The cell suspension was filtered through a 300-mesh filter into a 1.5 ml Eppendorf tube. Centrifuged for 5 minutes at 3000 rpm. The supernatant was discarded after centrifugation. Cells were resuspended in 50 μl of PBS (Biopico, China) containing 2% FBS. In the lymph node cell suspension, CD4-FITC (Rat anti-Mouse, Cat#553046, BD Biosciences, NJ, USA) and CD152-PE (Hamster anti-Mouse, Cat#553720, BD Biosciences, NJ, USA) diluted 1:10 with PBS were added, and the mixture was incubated on ice for 30 min. Cells were washed with PBS and centrifuged at 3000 rpm for 5 min. Cells were resuspended in 200 μl of PBS containing 2% FBS. 50,000 viable cells were analyzed using flow cytometry (Accuri C6Plus, BD Biosciences, NJ, USA).

[0159] 73. Result

[0160] result( Figure 7 The display shows that it has a sequence list. <400> The bifunctional oligonucleotide (ICO-3) of the sequence shown in Figure 2 can downregulate CTLA-4 in CD4+. + ICO-3 is expressed on T cells. Therefore, ICO-3 can significantly reduce CD4 expression. + T cells are in a state of continuous activation. CD4 cells are in this state. + T cells can assist tumor-specific CD8 cells. + T cells kill tumor cells.

[0161] Example 8: Inhibitory effect of bifunctional oligonucleotide (ICO-2) on tumor growth and CTILA4 expression on T cells in bilateral tumor-bearing mice.

[0162] 8.1. Materials

[0163] 8.1.1 Mice

[0164] C57BL / 6 mice (6-8 weeks old) were purchased from Yisi Laboratory Animal Technology Co., Ltd., Changchun, China. All mice were housed in an SPF (specific pathogen free) environment at the Jilin University Laboratory Animal Center, with free access to food and water. All animal experimental procedures involved in this study strictly followed the National Research Council's Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Ethics Committee of the School of Basic Medical Sciences, Jilin University.

[0165] 8.1.2. Oligonucleotides

[0166] ICO-2 as described in Example 1.

[0167] 8.1.2. B16 melanoma cells

[0168] B16 melanoma cells (B16 cells) derived from C57BL / 6 mice were obtained from the American Type Culture Collection. They were cultured in complete RPMI medium at 37°C with 5% CO2. 2 Cells were cultured in a constant temperature and humidity incubator at a controlled concentration. All cells were periodically tested and confirmed to be free of mycoplasma contamination.

[0169] 8.1.3. Cell Culture Medium

[0170] Complete RPMI medium (Gibco) containing 10% (v / v) fetal bovine serum, 100 IU penicillin / ml and 100 IU streptomycin / ml. Serum-free RPMI medium, without fetal bovine serum, containing 100 IU penicillin / ml and 100 IU streptomycin / ml. RPMI medium with 2% FBS, containing 2% (v / v) FBS, 100 IU penicillin / ml and 100 IU streptomycin / ml.

[0171] 8.2 Methods

[0172] B16 cells (5×10) 5 Mice were inoculated bilaterally in the medial thighs near the inguinal lymph node drainage area, followed by ICO-2 treatment. Starting on day 1 post-inoculation, ICO-2 was injected every other day at the right injection site, and PBS was injected at the left site, for a total of five times. Figure 8 ).

[0173] 8.3 Results

[0174] result( Figure 8 The results showed that on day 10 after B16 cell inoculation, the number of inguinal lymph node cells on the ICO-2 injection side was significantly increased, indicating that ICO-2 promoted and maintained lymphocyte activation and proliferation. This was accompanied by inhibition of melanoma growth on the ICO-2 injection side; ICO-2 increased CTLA-4 levels. + LNCs + CTLA-4 + CD4 + and CTLA-4 + CD8 + The percentage of cells, and reduced CTLA-4 in LNCs, CD4 + T cells and CD8 + ICO-2 expression on T cells suggests that it may maintain B16 cell-specific CD4 expression by inhibiting CTLA-4 expression. + T cells and CD8 + The sustained activation of T cells resulted in their ability to inhibit melanoma growth.

[0175] Example 9: Inhibitory effect of bifunctional oligonucleotide (ICO-2) on tumor growth in unilateral melanoma-bearing mice

[0176] 9.1. Materials

[0177] 9.1.1 Mice

[0178] C57BL / 6 mice (6-8 weeks old) were purchased from Yisi Laboratory Animal Technology Co., Ltd., Changchun, China. All mice were housed in an SPF (specific pathogen free) environment at the Jilin University Laboratory Animal Center, with free access to food and water. All animal experimental procedures involved in this study strictly followed the National Research Council's Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Ethics Committee of the School of Basic Medical Sciences, Jilin University.

[0179] 9.1.2. Oligonucleotides

[0180] ICO-2 as described in Example 1.

[0181] 9.1.3. B16 melanoma cells

[0182] B16 melanoma cells (B16 cells) derived from C57BL / 6 mice were obtained from the American Type Culture Collection. They were cultured in complete RPMI medium at 37°C with 5% CO2. 2 Cells were cultured in a constant temperature and humidity incubator at a controlled concentration. All cells were periodically tested and confirmed to be free of mycoplasma contamination.

[0183] 9.1.4. Cell Culture Medium

[0184] Complete RPMI medium (Gibco) containing 10% (v / v) fetal bovine serum, 100 IU penicillin / ml and 100 IU streptomycin / ml. Serum-free RPMI medium, without fetal bovine serum, containing 100 IU penicillin / ml and 100 IU streptomycin / ml. RPMI medium with 2% FBS, containing 2% (v / v) FBS, 100 IU penicillin / ml and 100 IU streptomycin / ml.

[0185] 9.2. Methods

[0186] C57BL / 6 mice were subcutaneously injected with 1×10⁻⁶ cells into the medial right thigh (near the inguinal lymph node drainage area). 5 B16 cells were injected subcutaneously into the right injection site on days 1, 3, 5, 7, and 9 post-inoculation with either 100 μl of PBS containing 20 μg ICO-2 or 100 μl of PBS. Tumor volume was measured and mortality was recorded starting on day 10. Tumor volume (mm) 3 = Length (diameter of the longest tumor in mm) × Width (diameter of the shortest tumor in mm) 2 ×0.52.

[0187] 9.3 Results

[0188] The results (Figure 1.9) show that, as in the sequence list <400> The bifunctional oligonucleotide (ICO-2) with the sequence shown in Figure 1 can inhibit the growth of melanoma and enhance anti-tumor immune memory.

[0189] Example 10: Inhibitory effect of bifunctional oligonucleotide (ICO-2) on tumor growth in mice bearing colon cancer cells.

[0190] 10.1. Materials

[0191] 10.1.1 Mice

[0192] Female BALB / c mice (6-8 weeks old) were purchased from Yisi Laboratory Animal Technology Co., Ltd., Changchun, China. All mice were housed in an SPF (specific pathogen free) environment at the Experimental Animal Center of Jilin University, with free access to food and water. All animal experimental procedures involved in this study strictly followed the National Research Council's Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Ethics Committee of the School of Basic Medical Sciences, Jilin University.

[0193] 10.1.2. Oligonucleotides

[0194] ICO-2 as described in Example 1.

[0195] 10.1.3. CT26 colon cancer cells

[0196] CT26 colon cancer cells (CT26 cells) derived from BALB / c mice were purchased from the American Type Culture Collection. They were cultured in complete RPMI medium at 37°C with 5% CO2. 2 Cells were cultured in a constant temperature and humidity incubator at a controlled concentration. All cells were periodically tested and confirmed to be free of mycoplasma contamination.

[0197] 10.1.4. Cell Culture Medium

[0198] Complete RPMI medium (Gibco) containing 10% (v / v) fetal bovine serum (FCS), 100 IU penicillin / ml and 100 IU streptomycin / ml. Serum-free RPMI medium, without fetal bovine serum, containing 100 IU penicillin / ml and 100 IU streptomycin / ml. RPMI medium with 2% FBS, containing 2% (v / v) FBS, 100 IU penicillin / ml and 100 IU streptomycin / ml.

[0199] 10.2 Methods

[0200] Subcutaneous injection of 5×10⁵ cells into the medial right thigh (near the inguinal lymph node drainage area) of BALB / C mice. 5 CT26 cells were injected subcutaneously into the right injection site on days 1, 3, 5, 7, and 9 post-inoculation with either 100 μl of PBS containing 20 μg ICO-2 or 100 μl of PBS. Tumor volume was measured starting on day 13, and mouse mortality was recorded. Tumor volume (mm)3 = Length (longest tumor diameter in mm) × Width (shortest tumor diameter in mm) 2 ×0.52.

[0201] 10.3 Results

[0202] result( Figure 10 The display shows that it has a sequence list. <400> The bifunctional oligonucleotide (ICO-2) with the sequence shown in Figure 1 can inhibit the growth of colon cancer and prolong the survival of colon cancer-bearing mice.

[0203] Example 11: Inhibitory effect of bifunctional oligonucleotide (ICO-2) on tumor growth formed by lung cancer cells.

[0204] 11.1 Materials

[0205] 11.1.1 Mice

[0206] C57BL / 6 mice (6-8 weeks old) were purchased from Yisi Laboratory Animal Technology Co., Ltd., Changchun, China. All mice were housed in an SPF (specific pathogen free) environment at the Experimental Animal Center of Jilin University, with free access to food and water. All animal experimental procedures involved in this study strictly followed the National Research Council's Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Ethics Committee of the School of Basic Medical Sciences, Jilin University.

[0207] 11.1.2. Oligonucleotides

[0208] ICO-2 as described in Example 1.

[0209] 11.1.3 Cells

[0210] Lewis lung carcinoma (LLC) cells (ATCC) were derived from C57BL / 6 mice. The cells were cultured in complete RPMI 1640 medium at 37°C and 5% CO2. Cells were regularly tested and confirmed to be free of mycoplasma contamination.

[0211] 11.1.4. Cell Culture Medium

[0212] Complete RPMI medium (Gibco) containing 10% (v / v) fetal bovine serum, 100 IU penicillin / ml and 100 IU streptomycin / ml. Serum-free RPMI medium, without fetal bovine serum, containing 100 IU penicillin / ml and 100 IU streptomycin / ml. RPMI medium with 2% FBS, containing 2% (v / v) FBS, 100 IU penicillin / ml and 100 IU streptomycin / ml.

[0213] 11.2. Methods

[0214] C57BL / 6 mice were subcutaneously injected with 1×10⁻⁶ cells into the medial right thigh (near the inguinal lymph node drainage area). 5 Lewis lung cancer cells (LLCC) were subcutaneously injected with 100 μl of PBS containing 20 μg ICO-2 or 100 μl of PBS at the right injection site on days 1, 3, 5, 7, and 9 post-inoculation. Tumor volume was measured starting on day 13. Tumor volume (mm) 3 = Length (diameter of the longest tumor in mm) × Width (diameter of the shortest tumor in mm) 2

[0215] ×0.52.

[0216] 11.3 Results

[0217] The results (Figure 11) show that, as in the sequence list <400> The bifunctional oligonucleotide (ICO-2) with the sequence shown in Figure 1 can inhibit the growth of lung cancer.

[0218] Example 12: Inhibitory effect of bifunctional oligonucleotide (ICO-3) on the growth of melanoma formed from B16 cells.

[0219] 12.1. Materials

[0220] 12.1.1. Mice

[0221] C57BL / 6 mice (6-8 weeks old) were purchased from Yisi Laboratory Animal Technology Co., Ltd., Changchun, China. All mice were housed in an SPF (specific pathogen free) environment at the Experimental Animal Center of Jilin University, with free access to food and water. All animal experimental procedures involved in this study strictly followed the National Research Council's Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Ethics Committee of the School of Basic Medical Sciences, Jilin University.

[0222] 12.1.2. Oligonucleotides

[0223] ICO-3 as described in Example 1.

[0224] 12.1.3. B16 melanoma cells

[0225] B16 melanoma cells (B16 cells) derived from C57BL / 6 mice were obtained from the American Type Culture Collection. They were cultured in complete RPMI medium at 37°C with 5% CO2. 2 Cells were cultured in a constant temperature and humidity incubator at a controlled concentration. All cells were periodically tested and confirmed to be free of mycoplasma contamination.

[0226] 12.1.4. Cell Culture Medium

[0227] Complete RPMI medium (Gibco) containing 10% (v / v) fetal bovine serum, 100 IU penicillin / ml and 100 IU streptomycin / ml. Serum-free RPMI medium, without fetal bovine serum, containing 100 IU penicillin / ml and 100 IU streptomycin / ml. RPMI medium with 2% FBS, containing 2% (v / v) FBS, 100 IU penicillin / ml and 100 IU streptomycin / ml.

[0228] 12.2. Methods

[0229] C57BL / 6 mice were subcutaneously injected with 1×10⁻⁶ cells into the medial right thigh (near the inguinal lymph node drainage area). 5 B16 cells were injected subcutaneously into the right injection site on days 1, 3, 5, 7, and 9 post-inoculation with either 100 μl of PBS containing 20 μg ICO-3 or 100 μl of PBS. Tumor volume was measured and mortality was recorded starting on day 10. Tumor volume (mm) 3= Length (diameter of the longest tumor in mm) × Width (diameter of the shortest tumor in mm) 2 ×0.52.

[0230] 12.3. Results

[0231] result( Figure 12 The display shows that it has a sequence list. <400> The bifunctional oligonucleotide (ICO-3) with the sequence shown in Figure 2 can inhibit the growth of melanoma.

[0232] Example 13: Inhibitory effect of bifunctional oligonucleotide (ICO-3) on tumor growth in mice bearing colon cancer cells.

[0233] 13.1. Materials

[0234] 13.1.1 Mice

[0235] Female BALB / c mice (6-8 weeks old) were purchased from Yisi Laboratory Animal Technology Co., Ltd., Changchun, China. All mice were housed in an SPF (specific pathogen free) environment at the Experimental Animal Center of Jilin University, with free access to food and water. All animal experimental procedures involved in this study strictly followed the National Research Council's Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Ethics Committee of the School of Basic Medical Sciences, Jilin University.

[0236] 13.1.2. Oligonucleotides

[0237] ICO-3 as described in Example 1.

[0238] 13.1.3. CT26 colon cancer cells

[0239] CT26 colon cancer cells (CT26 cells) derived from BALB / c mice were purchased from the American Type Culture Collection. They were cultured in complete RPMI medium at 37°C with 5% CO2. 2 Cells were cultured in a constant temperature and humidity incubator at a controlled concentration. All cells were periodically tested and confirmed to be free of mycoplasma contamination.

[0240] 13.1.4. Cell Culture Medium

[0241] Complete RPMI medium (Gibco) containing 10% (v / v) fetal bovine serum, 100 IU penicillin / ml and 100 IU streptomycin / ml. Serum-free RPMI medium, without fetal bovine serum, containing 100 IU penicillin / ml and 100 IU streptomycin / ml. RPMI medium with 2% FBS, containing 2% (v / v) FBS, 100 IU penicillin / ml and 100 IU streptomycin / ml.

[0242] 13.2 Methods

[0243] Subcutaneous injection of 5×10⁵ cells into the medial right thigh (near the inguinal lymph node drainage area) of BALB / C mice. 5 CT26 cells were injected subcutaneously into the right injection site on days 1, 3, 5, 7, and 9 post-inoculation with either 100 μl of PBS containing 20 μg ICO-3 or 100 μl of PBS. Tumor volume was measured and mortality was recorded starting on day 13. Tumor volume (mm) 3 = Length (diameter of the longest tumor in mm) × Width (diameter of the shortest tumor in mm) 2 ×0.52.

[0244] 13.3 Results

[0245] result( Figure 13 Studies have shown that a 20 μg dose of ICO-3 can significantly reduce the volume of tumors formed from CT26 colon cancer cells.

[0246] Example 14: Inhibitory effect of bifunctional oligonucleotide (ICO-3) on tumor growth caused by lung cancer cells.

[0247] 14.1 Materials

[0248] 14.1.1 Mice

[0249] C57BL / 6 mice (6-8 weeks old) were purchased from Yisi Laboratory Animal Technology Co., Ltd., Changchun, China. All mice were housed in an SPF (specific pathogen free) environment at the Experimental Animal Center of Jilin University, with free access to food and water. All animal experimental procedures involved in this study strictly followed the National Research Council's Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Ethics Committee of the School of Basic Medical Sciences, Jilin University.

[0250] 14.1.2. Oligonucleotides

[0251] ICO-3 as described in Example 1.

[0252] 14.1.3 Cells

[0253] Lewis lung carcinoma (LLC) cells (ATCC) were derived from C57BL / 6 mice. The cells were cultured in complete RPMI 1640 medium at 37°C and 5% CO2. Cells were regularly tested and confirmed to be free of mycoplasma contamination.

[0254] 14.1.4. Cell Culture Medium

[0255] Complete RPMI medium (Gibco) containing 10% (v / v) fetal bovine serum, 100 IU penicillin / ml and 100 IU streptomycin / ml. Serum-free RPMI medium, without fetal bovine serum, containing 100 IU penicillin / ml and 100 IU streptomycin / ml. RPMI medium with 2% FBS, containing 2% (v / v) FBS, 100 IU penicillin / ml and 100 IU streptomycin / ml.

[0256] 14.2. Methods

[0257] C57BL / 6 mice were subcutaneously injected with 1×10⁻⁶ cells into the medial right thigh (near the inguinal lymph node drainage area). 5 Lewis lung cancer cells (LLCC) were subcutaneously injected with 100 μl of PBS containing 20 μg ICO-3 or 100 μl of PBS at the right injection site on days 1, 3, 5, 7, and 9 post-inoculation. Tumor volume was measured starting on day 13. Tumor volume (mm) 3 = Length (diameter of the longest tumor in mm) × Width (diameter of the shortest tumor in mm) 2 ×0.52.

[0258] 14.3 Results

[0259] result( Figure 14 The display shows that it has a sequence list. <400> The bifunctional oligonucleotide (ICO-3) with the sequence shown in Figure 2 can inhibit the growth of lung cancer.

Claims

1. Single-stranded deoxy oligonucleotides with a fully thiolated backbone as shown in SEQ ID NO: 1 and SEQ ID NO:

2.

2. The single-stranded deoxy oligonucleotides according to claim 1 may be chemically modified.

3. The single-chain deoxy oligonucleotides according to claims 1 and 2 have TLR9 agonist activity and can stimulate B lymphocytes and dendritic cells to express co-stimulatory molecules, thus producing anti-tumor effects by enhancing the second activation signal of anti-tumor T cells.

4. The single-stranded deoxyoligonucleotides according to claims 1 and 2, which can inhibit CD4 + T cells and CD8 + T cells express cytotoxic T lymphocyte-associated protein 4 (CTLA-4), and therefore can maintain CD4+. + T cells and CD8 + The powerful activation of T cells produces an anti-tumor effect.

5. The single-chain deoxy oligonucleotides according to claims 1 and 2 can be used alone for individual tumor treatment.

6. The single-chain deoxy oligonucleotides according to claims 1 and 2 can be used in combination with antitumor agents for individual tumor treatment.

7. The single-chain deoxy oligonucleotides according to claims 1 and 2 can work in combination with other antitumor agents to produce a synthetic lethal effect on tumor cells in an individual.

8. The single-chain deoxy oligonucleotides according to claims 1 and 2 can be used in combination with tumor vaccines to exert a tumor therapeutic effect.

9. The single-chain deoxy oligonucleotides according to claims 1 and 2 can be used in combination with other tumor vaccine adjuvants to exert a tumor therapeutic effect.

10. The single-stranded deoxy oligonucleotides according to claims 1 and 2 can be used as adjuvants for tumor neoantigens.

11. The single-stranded deoxy oligonucleotides according to claims 1 and 2, which can be used in individual treatment of melanoma.

12. The single-chain deoxy oligonucleotides according to claims 1 and 2 can be used in combination with other antitumor agents for individual treatment of melanoma.

13. The single-chain deoxy oligonucleotides according to claims 1 and 2 can be used in the individual treatment of colon cancer.

14. The single-chain deoxy oligonucleotides according to claims 1 and 2 can be used in combination with other antitumor agents for the individual treatment of colon cancer.

15. The single-stranded deoxy oligonucleotides according to claims 1 and 2 can be used in the individual treatment of lung cancer.

16. The single-chain deoxy oligonucleotides according to claims 1 and 2 can be used in combination with other antitumor agents for individual treatment of lung cancer.

17. The single-stranded deoxy oligonucleotides according to claims 1 and 2, which can be delivered using a delivery system to treat tumors.

18. The single-stranded deoxy oligonucleotides according to claims 1 and 2, which can be used to treat tumors via local application routes including but not limited to intratumoral, peritumoral, tumor drainage lymph nodes, subcutaneous or intramuscular routes.

19. The single-chain deoxy oligonucleotides according to claims 1 and 2 are non-antibody immune checkpoint inhibitors that do not induce complement-dependent cytotoxicity or antibody-dependent cell-mediated cytotoxicity caused by immune checkpoint antibodies, thus avoiding immune-related adverse events caused by such toxicity.

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