JP2019531095A - Immunosuppressive reversion oligonucleotide that inhibits expression of CD73 - Google Patents

Abstract

The present invention relates to an immunosuppressive reversion oligonucleotide comprising 12 to 18 nucleotides, wherein at least one of the nucleotides is modified, and the oligonucleotide is a sequence of SEQ ID NO: 1 (human) and / or SEQ ID NO: 2 (mouse) Reference is made to oligonucleotides that hybridize with the nucleic acid sequence of the extracellular enzyme of (CD73) and inhibit at least 50% of CD73 expression. The present invention is further directed to pharmaceutical compositions comprising such oligonucleotides.

Description

  The present disclosure relates to an immunosuppression reversion oligonucleotide that hybridizes with the nucleic acid sequence of an extracellular enzyme (ectoenzye: ectoenzyme, ecto-type enzyme) (NT5E or CD73), such an immunosuppression reversion oligonucleotide, and pharmaceutically acceptable And a pharmaceutical composition comprising a carrier, excipient and / or diluent.

  In recent years, the treatment of several different diseases such as malignant tumors has been very successful by applying immunotherapy, in particular by inhibitors called “immune checkpoints”. Such checkpoints are molecules of the immune system that enhance (costimulatory molecules) or weaken the signal. The concept of therapeutic approach is based on the activation of an endogenous anti-tumor immune response. For example, many cancers protect themselves from the immune system by inhibiting the activity of T cells and NK cells, respectively. For example, immune checkpoint modulators, i.e. stimulators or inhibitors are CTLA-4, PD-1, PD-L1, LAG-3, VISTA, A2AR, BTLA, IDO, CD39, CD73, STAT3, TDO2, TIM-3 , MICA, NKG2A, KIR, TIGIT, TGF-beta, Ox40, GITR, CD27, CD160, 2B4 and 4-1BB are targeted.

CD73 needs to be considered as one new and promising candidate for improving immunity against various types of cancer. CD73 is an extracellular enzyme (NTPdase) that catalyzes the conversion of AMP to immunosuppressive adenosine. CD73 cooperates with CD39, known to convert ATP to ADP and ADP to AMP, but acts downstream of CD39. Adenosine, adenosine receptors A1, adenosine receptor A _2A, via adenosine receptors A _2B and adenosine receptor A ₃ exerts its effect. The range of immune cells that express adenosine receptors and are potentially affected by the immunomodulatory action of adenosine are T lymphocytes, natural killer (NK) cells, NKT cells, macrophages, DCs, neutrophils, masts Includes cells and B cells.

  CD73 is found in most tissues and many cell types, including a subset of lymphocytes, macrophages, dendritic cells, endothelial cells and epithelial cells. Hypoxia induces an increase in CD73 mRNA, protein expression and CD73 activity in mouse microvascular endothelial cells. In particular, CD73 is highly expressed in many different human (solid and blood) tumors, and increased expression and activity are associated with tumor invasion and metastasis, and reduced patient survival. CD73 RNA expression and enzyme activity varies in various breast cancer cell lines.

Dying cancer cells release ATP into the extracellular space within the tumor microenvironment. Live tumor cells often express high levels of CD39 and CD73, converting ATP to immunosuppressive adenosine. This allows tumor cells to grow and metastasize uncontrolled. By binding to A _2A or A _2B receptors on lymphocytes, adenosine mediates immunosuppressive signals for such cells. For example, T cells are inhibited in their proliferation, production and activity of cytotoxic cytokines. NK cells show reduced cytotoxic potential. When adenosine induces alternative activity of macrophages (immunosuppressive M2 phenotype), production of inflammatory cytokines is reduced, but production of immunosuppressive cytokine IL-10 is increased. The important role of CD73 as a suitable therapeutic target in various tumors is highlighted by the fact that tumor models using CD73 or A ₂ A receptor knockout mice show improved disease outcome.

  Anti-human CD73 monoclonal antibodies, such as Innate Pharma's anti-CD73 antibody (eg, Innate Pharma Poster # iph_poster_aarc2016_cd73) are currently under preclinical research in immunooncology cell-based assays. However, due to steric hindrance, monoclonal antibodies against CD73 may not be able to localize to the tumor microenvironment. Further, non-hydrolyzable small molecule inhibitors of CD73, such as AMPCP (adenosine 5 ′-(α, β-methylene) diphosphate that competitively inhibits CD73 activity; for example, Structure 20, pages 2161-2173, (2012, December 5) ADP analogs have been tested in animal models in vitro and in vivo, but require repeated administration at relatively high concentrations to successfully block CD73 enzyme activity It is.

  Immunotherapy results in long-term remission, but so far only in small patient groups. The reason for this may be that numerous immune checkpoints and optionally further immunosuppressive mechanisms are involved, for example, in the interaction between the immune system and tumor cells. The combination of immune checkpoints and potential other mechanisms can vary depending on the tumor evading biodefense and the individual condition of the subject.

  For the inhibition of some immunosuppressive mechanisms, the general approach using antibodies and / or small molecules is unsuitable or nearly unsuitable because the molecular target is located intracellularly or has no enzymatic activity . Thus, agents that are safe and effective in inhibiting "immune checkpoint" function, such as CD73, can be an important addition to the treatment of patients suffering from diseases or conditions affected by this enzyme activity, for example. .

The oligonucleotides of the present invention are very successful in inhibiting CD73 expression and activity, respectively. The mechanism of action of oligonucleotides is different from that of antibodies or small molecules,
(i) infiltration of solid tumor into tumor tissue,
(ii) blocking each of the multiple functions and activities of the target;
(iii) oligonucleotides together, or a combination of oligonucleotides and antibodies or small molecules, and
(iv) It is very advantageous with respect to inhibiting intracellular effects where the antibody is not accessible or cannot be inhibited by small molecules.

WO2014154843 A1

Structure 20, 2161-2173, 2012, December 5 Chackalamannil, Rotella, Ward, Comprehensive Modicinal Chemistry III Elsevier, 03.06.2017 Zhang et al., Gene Therapy, 2011, 18, 326-333. Stanton et al., Nucleic Acid Therapeutics, Vol. 22, No. 5, 2012

  Therefore, targeting CD73 expression on cancer and immune cells at the mRNA level with antisense oligonucleotides is a promising state-of-the-art technique for developing and improving immunotherapy against, for example, various cancers and immune diseases, respectively. It is.

  The present invention refers to an oligonucleotide, such as an immunosuppressive reversion oligonucleotide, comprising about 10-20 nucleotides, wherein at least one of the nucleotides has been modified. The oligonucleotide hybridizes, for example, to the nucleic acid sequence of the extracellular enzyme CD73 of SEQ ID NO: 1 (human) and / or the sequence of SEQ ID NO: 2 (mouse). The modified nucleotide is, for example, selected from the group consisting of a cross-linked nucleic acid (eg, LNA, cET, ENA, 2′fluoro modified nucleotide or 2′O-methyl modified nucleotide). In some embodiments, the oligonucleotide inhibits at least 50% of CD73 expression, and in some embodiments, the oligonucleotide inhibits CD73 expression at nanomolar concentrations.

  Antisense oligonucleotides have significant advantages over RNAi. Antisense oligonucleotides can be transfected in vitro without transfection reagents, and therefore this transfection is more in vivo than transfection using transfection reagents that are essential for RNAi transfection. Close to conditions. In vivo systemic administration of antisense oligonucleotides is possible in a variety of tissues, but in vivo administration of RNAi depends on the delivery system, eg, GalNAc in the liver. Furthermore, antisense oligonucleotides are shorter than RNAi and are therefore less complex to synthesize and take up into cells. RNAi regularly shows off-target effects of passenger strands that can initiate RNAi as well. The RISC loading of the passenger strand is an important issue for RNAi drugs because it targets RNAi activity on the unintended target of the passenger strand and can cause toxic side effects (Chackalamannil, Rotella, Ward, Comprehensive Modicinal Chemistry III Elsevier, see 03.06.2017). Antisense oligonucleotides do not contain passenger strands.

  The present invention further relates to pharmaceutical compositions comprising the immunosuppressive reverting oligonucleotides of the present invention, and optionally pharmaceutically acceptable carriers, excipients and / or diluents. In some embodiments, the pharmaceutical composition further comprises a chemotherapeutic agent, such as a platinum or gemcitabine, another oligonucleotide, an antibody and / or a small molecule that is effective, for example, in tumor therapy.

  In some embodiments, an oligonucleotide of the invention is a combination with another oligonucleotide, antibody and / or small molecule. Any of each such compound is isolated or combined in a pharmaceutical composition, where oligonucleotides, antibodies and / or small molecules are immunosuppressive factors such as IDO1, IDO2, CTLA-4, PD -1, PD-L1, LAG-3, VISTA, A2AR, CD39, CD73, STAT3, TDO2, TIM-3, TIGIT, TGF-beta, BTLA, MICA, NKG2A, KIR, CD160, Chop and / or Xbp1 inhibited Or irritate. In addition or alternatively, oligonucleotides, antibodies and / or small molecules inhibit or stimulate immunostimulatory factors such as 4-1BB, Ox40, KIR, GITR, CD27 and / or 2B4.

  Furthermore, the present invention relates to the use of an oligonucleotide or pharmaceutical composition of the invention in a method for preventing and / or treating disorders involving CD73 imbalance. In some embodiments, the disorder is, for example, an autoimmune disorder such as autoimmune arthritis or an autoimmune gastrointestinal disease such as inflammatory bowel disease (IBD) or colitis, an immune disorder such as a chronic viral infection such as HIV. Immune exhaustion due to infection, cardiovascular disorders, inflammatory disorders such as chronic respiratory tract inflammation, bacterial, viral and / or fungal infections such as sepsis or Mycobacterium bovis infection, liver disorders, chronic kidney disorders, Mental disorder and / or cancer. CD73 has many physiological roles, including regulation of barrier function, adaptation to hypoxia, ischemic preconditioning, anti-inflammation, and leukocyte extravasation. CD73 expression and activity on cancer cells is associated with poor prognosis and may promote metastasis. CD73 promotes human breast cancer cell adhesion, migration, invasion, and glioblastoma cell proliferation, and such processes depend on the production of adenosine by the enzyme. In some embodiments, the oligonucleotides or pharmaceutical compositions of the invention are administered, for example, locally or systemically.

  All documents cited or referred to in this specification ("documents cited in this specification") and all documents cited or referenced in documents cited in this specification are referred to in this specification or in this specification. Together with any manufacturer's instructions, descriptions, product specifications, and product descriptions for any product mentioned in the literature incorporated by reference, thereby incorporated herein by reference and used in the practice of the invention. Can be. More specifically, all of the documents referred to are incorporated by reference in the same way that each individual document is specifically and individually indicated to be incorporated by reference.

It is a figure which shows distribution of the human (h) CD73 antisense oligonucleotide couple | bonded with the site | part on hCD73 mRNA of sequence number 1 (NM_002526.3), these modifications, and length. The hCD73 antisense oligonucleotide was aligned to the hCD73 mRNA sequence of SEQ ID NO: 1. Different gray scales indicate different LNA modifications, and each symbol represents a different length of antisense oligonucleotide. It is a graph which shows the hCD73 mRNA knockdown efficacy of hmCD73 antisense oligonucleotide in human cancer cell line A-172 (human glioblastoma) in the first screening round. A-172 cells were treated with 10 μM of each antisense oligonucleotide for 3 days. As a control, cells were treated with neg1 (described in WO2014154843 A1), an antisense oligonucleotide having the sequence CGTTTAGGCTATGTACTT. Shown is residual human or mouse CD73 mRNA expression compared to untreated cells. Expression values were normalized to that of the housekeeping gene GAPDH. Shown is the mean +/- SD of triplicate wells. FIG. 6 is a graph showing the hCD73 mRNA knockdown efficacy of hmCD73 antisense oligonucleotide in human cancer cell line A-172 (human glioblastoma) in the second screening round (Part 1). A-172 cells were treated with 10 μM of each antisense oligonucleotide for 3 days. As a control, cells were treated with neg1 (described in WO2014154843 A1), an antisense oligonucleotide having the sequence CGTTTAGGCTATGTACTT. Shown is residual human or mouse CD73 mRNA expression compared to untreated cells. Expression values were normalized to that of the housekeeping gene GAPDH. Shown is the mean +/- SD of triplicate wells. FIG. 10 is a graph showing the hCD73 mRNA knockdown efficacy of hmCD73 antisense oligonucleotide in human cancer cell line A-172 (human glioblastoma) in the second screening round (Part 2). A-172 cells were treated with 10 μM of each antisense oligonucleotide for 3 days. As a control, cells were treated with neg1 (described in WO2014154843 A1), an antisense oligonucleotide having the sequence CGTTTAGGCTATGTACTT. Shown is residual human or mouse CD73 mRNA expression compared to untreated cells. Expression values were normalized to that of the housekeeping gene GAPDH. Shown is the mean +/- SD of triplicate wells. FIG. 2 is a graph showing hCD73 mRNA knockdown efficacy of hmCD73 antisense oligonucleotide in EFO-21 (human ovarian cystadenocarcinoma) in one screening round (Part 1). EFO-21 cells were treated with 10 μM of each antisense oligonucleotide for 3 days. As a control, cells were treated with neg1 (described in WO2014154843 A1), an antisense oligonucleotide having the sequence CGTTTAGGCTATGTACTT. Shown is residual human or mouse CD73 mRNA expression compared to untreated cells. Expression values were normalized to that of the housekeeping gene HPRT1. Mean +/- SD of 3 wells is shown. FIG. 3 is a graph showing the hCD73 mRNA knockdown efficacy of hmCD73 antisense oligonucleotides in EFO-21 (human ovarian cystadenocarcinoma) in one screening round (Part 2). EFO-21 cells were treated with 10 μM of each antisense oligonucleotide for 3 days. As a control, cells were treated with neg1 (described in WO2014154843 A1), an antisense oligonucleotide having the sequence CGTTTAGGCTATGTACTT. Shown is residual human or mouse CD73 mRNA expression compared to untreated cells. Expression values were normalized to that of the housekeeping gene HPRT1. Shown is the mean +/- SD of triplicate wells. FIG. 2 is a graph showing hCD73 mRNA knockdown efficacy of hmCD73 antisense oligonucleotide in one mouse cell line 4T1 (breast cancer) in one screening round (Part 1). 4T1 cells were treated with 10 μM of each antisense oligonucleotide for 3 days. As a control, cells were treated with neg1 (described in WO2014154843 A1), an antisense oligonucleotide having the sequence CGTTTAGGCTATGTACTT. Shown is residual human or mouse CD73 mRNA expression compared to untreated cells. Expression values were normalized to that of the housekeeping gene HPRT1. Shown is the mean +/- SD of triplicate wells. FIG. 3 is a graph showing hCD73 mRNA knockdown efficacy of hmCD73 antisense oligonucleotide in mouse cell line 4T1 (breast cancer) in one screening round (Part 2). 4T1 cells were treated with 10 μM of each antisense oligonucleotide for 3 days. As a control, cells were treated with neg1 (described in WO2014154843 A1), an antisense oligonucleotide having the sequence CGTTTAGGCTATGTACTT. Shown is residual human or mouse CD73 mRNA expression compared to untreated cells. Expression values were normalized to that of the housekeeping gene HPRT1. Shown is the mean +/- SD of triplicate wells. FIG. 6 is a graph showing the hCD73 mRNA knockdown efficacy of hmCD73 antisense oligonucleotides in human SKOV-3 (human ovarian adenocarcinoma cancer) cells in one screening round. SKOV-3 cells were treated with 10 μM of each antisense oligonucleotide for 3 days. As a control, cells were treated with neg1 (described in WO2014154843 A1), an antisense oligonucleotide having the sequence CGTTTAGGCTATGTACTT. Shown is residual human or mouse CD73 mRNA expression compared to untreated cells. Expression values were normalized to that of the housekeeping gene HPRT1. Average +/- SD of triplicate wells is shown. FIG. 6 is a graph showing a correlation analysis of hmCD73 antisense oligonucleotide potency in human EFO-21 cells compared to human A-172 cells. FIG. 6 is a graph showing a correlation analysis of hmCD73 antisense oligonucleotide potency in human A-172 cells compared to mouse 4T1 cells. With selected hmCD73 antisense oligonucleotides (A05008HM (SEQ ID NO: 4), A05009HM (SEQ ID NO: 6), A05018HM (SEQ ID NO: 3), A05026HM (SEQ ID NO: 8), and A05028HM (SEQ ID NO: 5)) in SKOV-3 cells) It is a graph which shows concentration-dependent hmCD73 mRNA knockdown. SKOV-3 cells were treated with the indicated concentrations of each antisense oligonucleotide for 3 days. Residual hCD73 expression is shown relative to untreated control cells (set as 100). hCD73 mRNA expression values were normalized to the expression of the housekeeping gene HPRT1. Shown is the mean +/- SD of triplicate wells. Concentration-dependent target knockdown was used to calculate the IC ₅₀ values shown in Table 7 for SKOV-3 cells. Selected hmCD73 antisense oligonucleotides in EFO-21 (human ovarian cancer) (A05018HM (SEQ ID NO: 3), A05027HM (SEQ ID NO: 30), A05028HM (SEQ ID NO: 5), and A05037HM (SEQ ID NO: 39 in EFO-21 cells) FIG. 6 is a graph showing concentration-dependent hmCD73 mRNA knockdown according to FIG. EFO-21 cells were treated with the indicated concentrations of each antisense oligonucleotide for 3 days. Residual hCD73 expression is shown relative to untreated control cells (set as 100). hCD73 mRNA expression values were normalized to the expression of the housekeeping gene HPRT1. Shown is the mean +/- SD of triplicate wells. Concentration-dependent target knockdown was used to calculate the IC ₅₀ values shown in Table 8 for EFO-21 cells. It is a graph which shows the hmCD73 mRNA knockdown efficacy of the hmCD73 antisense oligonucleotide in human and mouse cancer cell lines. EFO-21 (human ovarian cystadenocarcinoma) cells were treated with 10 μM of each antisense oligonucleotide for 3 days. Shown is residual hmCD73 mRNA expression compared to untreated cells (set as 1). Expression values were normalized to the expression of the housekeeping gene HPRT1. Shown is the mean +/- SD of triplicate wells. It is a graph which shows the hmCD73 mRNA knockdown efficacy of the hmCD73 antisense oligonucleotide in human and mouse cancer cell lines. SKOV-3 (human ovarian adenocarcinoma) cells were treated with 10 μM of each antisense oligonucleotide for 3 days. Shown is residual hmCD73 mRNA expression compared to untreated cells (set as 1). Expression values were normalized to the expression of the housekeeping gene HPRT1. Shown is the mean +/- SD of triplicate wells. It is a graph which shows the hmCD73 mRNA knockdown efficacy of the hmCD73 antisense oligonucleotide in human and mouse cancer cell lines. 4T1 (mouse breast cancer) cells were treated with 10 μM of each antisense oligonucleotide for 3 days. Shown is residual hmCD73 mRNA expression compared to untreated cells (set as 1). Expression values were normalized to the expression of the housekeeping gene HPRT1. Shown is the mean +/- SD of triplicate wells. FIG. 5 is a graph showing EFO-21 cells treated with the indicated concentrations of each antisense oligonucleotide for 3 days. hCD73 mRNA expression values were normalized to the expression of the housekeeping gene HPRT1. Residual hCD73 mRNA expression compared to untreated cells (set as 100) is shown. Shown is the mean +/- SD of triplicate wells. 7A and 7B are graphs showing concentration-dependent hCD73 mRNA and protein knockdown by A05008HM (SEQ ID NO: 4), A05018HM (SEQ ID NO: 3) and A05028HM (SEQ ID NO: 5). Analysis of protein expression and cell viability by flow cytometry of 7-AAD staining of CD73 (FIG. 7A) and SKOV-3 cells (FIG. 7B) was performed for 6 days after treatment with the indicated antisense oligonucleotide. As a control, cells were similarly treated with neg1. The median fluorescence intensity of CD73 protein expression (FIG. 7A) and total dead cells (7-ADD positive cells) (FIG. 7B) compared to the number of control cells not treated with any antisense oligonucleotide (= 1) is shown. FIG. 5 is a graph showing knockdown of CD73 protein expression on SKOV3 and EFO-21 cells after antisense oligonucleotide treatment. CD73 protein expression on both test cell lines was analyzed for 6 days after treatment with A05018HM (SEQ ID NO: 3). As a control, cells were similarly treated with neg1. The median fluorescence intensity of CD73 protein expression compared to the expression of control cells not treated with any antisense oligonucleotide (= 1) is shown. 9A-9D are graphs showing the effect of hCD73 knockdown on extracellular pyrophosphate levels and cell viability by SKOV-3 and EFO-21 cells. Analyzing pyrophosphate production as an indirect measure of adenosine production in cell supernatants from EFO-21 cells (Figure 9A) and SKOV-3 cells (Figure 9C) treated with A05018HM (SEQ ID NO: 3) for 6 days did. Prior to measurement, 500 μM exogenous AMP was added to the cells at the indicated time points. The effect of treatment with A05018HM (SEQ ID NO: 3) on cell viability of EFO-21 cells (FIG. 9B) and SKOV-3 cells (FIG. 9D) was tested using the cell titer blue assay. FIGS. 10A-10C are graphs showing EFO-21 cells treated with 5 μM CD73-specific antisense oligonucleotide A05018HM (black bar graph) or control oligonucleotide S6 (white bar graph) for a total treatment time of 6 days. . hCD73 protein expression was analyzed by flow cytometry. FIG. 10A shows residual hCD73 expression compared to untreated cells (dot bar graph; set as 1). To analyze the ability to degrade extracellular AMP, 300 μM AMP was added to cells or cell-free PBS (striped bar graph) 6.5 hours before completion. FIG. 10B shows relative AMP levels compared to cell-free AMP-added PBS (set as 1), and FIG. 10C shows the absolute concentration of adenosine in the cell supernatant (cell-conditioned PBS). Average values of 3 wells +/- SD or single values of conditions with cell-free PBS are shown. Human CD4 ⁺ labeled with cell growth dye, activated with anti-CD3 and treated with 5 μM CD73-specific antisense oligonucleotide A05018HM (black bar graph) or control oligonucleotide S6 (white bar graph) for a total treatment time of 5 days It is a graph which shows a T cell. In the vehicle control (striped bar graph), cells were activated with anti-CD3 alone. Subsequently, 300 μM AMP or vehicle was added to the cells on days 3 and 4 after the start of oligonucleotide treatment. FIG. 11A shows CD73 protein expression of CD4 ⁺ T cells analyzed using flow cytometry 5 days after the start of oligonucleotide treatment. Shown is the mean +/- SD of triplicate wells. Human CD4 ⁺ labeled with cell growth dye, activated with anti-CD3 and treated with 5 μM CD73-specific antisense oligonucleotide A05018HM (black bar graph) or control oligonucleotide S6 (white bar graph) for a total treatment time of 5 days It is a graph which shows a T cell. In the vehicle control (striped bar graph), cells were activated with anti-CD3 alone. Subsequently, 300 μM AMP or vehicle was added to the cells on days 3 and 4 after the start of oligonucleotide treatment. FIG. 11B shows the proliferation of CD4 ⁺ T cells analyzed using flow cytometry 5 days after the start of oligonucleotide treatment. Shown is the mean +/- SD of triplicate wells. Labeled with cell proliferation dye, activated with anti-CD3, human treated during the whole treatment time of 5 days at 5μM of CD73-specific antisense oligonucleotide A05018HM (black bars) or control oligonucleotide S6 (white bars) CD4 ⁺ It is a graph which shows a T cell. In the vehicle control (striped bar graph), cells were activated with anti-CD3 alone. Subsequently, 300 μM AMP or vehicle was added to the cells on days 3 and 4 after the start of oligonucleotide treatment. FIG. 11C shows the absolute cell number of CD4 ⁺ T cells analyzed using flow cytometry 5 days after the start of oligonucleotide treatment. Shown is the mean +/- SD of triplicate wells. It is a graph which shows the in vivo effect | action of hmCD73 antisense oligonucleotide (A05027HM) treatment with respect to the expression of mCD73 mRNA in a mouse | mouth liver. The results shown in FIG. 12 show mCD73 mRNA levels in the liver of A05027HM- or vehicle-treated (white bar graph) mice. This is a sequence showing hCD73 mRNA of SEQ ID NO: 1 (NM_002526.3).

  The present invention provides for the first time human and mouse oligonucleotides that hybridize with the mRNA sequence of ectonucleotidase CD73 and inhibit, for example, CD73 expression and activity, respectively, on tumor cells or tumor-associated immune cells. As a result, the level of ATP increases and the levels of its degradation products, such as ADP, AMP and immunosuppressive adenosine, decrease. All of these effects result in increased anti-tumor immune cells, immune activation (eg, via cytotoxic T cells or NK cells) and tumor cell recognition and elimination, respectively. Thus, the oligonucleotides of the invention represent an interesting and highly effective tool for use in methods of preventing and / or treating disorders with increased CD73 expression and activity, respectively.

  In the following, the elements of the invention are described in more detail. Although such elements are listed with particular embodiments, it should be understood that they can be combined in any manner and in any number to create further embodiments. The variously described examples and embodiments should not be construed to limit the invention to only the explicitly described embodiments. This description should be understood to assist and encompass embodiments in which the explicitly described embodiments are combined with any number of the disclosed elements. Further, any permutations and combinations of all elements described in this application should be considered disclosed by the description of this application, unless the context indicates otherwise.

  Throughout the specification and claims, unless the context demands otherwise, the words “comprise” and variations such as “comprises” and “comprising” are members described. , An integer or step, or a member, an integer or a group of steps, but is not meant to exclude any other member, integer or step, or a group of members, integers or steps Is done. The terms `` one (a) '' and `` one '' and `` the '' and similar references used in connection with the description of the invention (especially in the context of the claims) are hereby incorporated by reference. Unless otherwise indicated in context, or unless the context explicitly contradicts, it should be construed to include both singular and plural. The recitation of value ranges herein is intended only to serve as a concise way of referring individually to each individual value within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually listed herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary languages provided herein (e.g., "such as", "for example") are merely intended to better illustrate the invention. Unless otherwise stated, no limitation of the scope of the invention is posed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

  The oligonucleotide of the present invention is an antisense oligonucleotide consisting of or including, for example, 10 to 25 nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 12 to 18 nucleotides, or 14 to 17 nucleotides. The oligonucleotide consists of or comprises, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 nucleotides. The oligonucleotides of the present invention comprise at least one modified nucleotide. Modified nucleotides are, for example, bridged nucleotides such as locked nucleic acids (LNA, eg 2 ′, 4′-LNA), cET, ENA, 2 ′ fluoro modified nucleotides, 2 ′ O-methyl modified nucleotides or combinations thereof. In some embodiments, the oligonucleotides of the invention comprise nucleotides with the same or different modifications. In some embodiments, the oligonucleotides of the invention comprise a modified phosphate backbone, wherein the phosphate is, for example, phosphorothioate or methylphosphonic acid or both.

  The oligonucleotide of the present invention comprises one or more modified nucleotides at the 3 ′ and / or 5 ′ end of the oligonucleotide and / or at any position within the oligonucleotide, wherein the modified nucleotide is 1, 2, Three, four, five or six modified nucleotides are contiguous or the modified nucleotide is combined with one or more unmodified nucleotides. The following Table 1 shows embodiments of oligonucleotides comprising modified nucleotides, such as LNA indicated by (+) and phosphorothioate (PTO) indicated by (*). Oligonucleotides consisting of or containing the sequences of Table 1 can include any other modified nucleotides and any other combination of modified and unmodified nucleotides. The oligonucleotides in Table 1 hybridize to human and mouse CD73 mRNA.

  The oligonucleotide of the present invention hybridizes with, for example, human or mouse CD73 mRNA of SEQ ID NO: 1 and / or SEQ ID NO: 2. Such oligonucleotides are called CD73 antisense oligonucleotides.

  In some embodiments, the oligonucleotide of the invention has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% of CD73 expression, such as human or mouse CD73, Inhibits 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Accordingly, the oligonucleotide of the present invention is an immunosuppression reversion oligonucleotide that reverts immunosuppression in, for example, a cell, tissue, organ, or subject. The oligonucleotides of the present invention can be used in nanomolar or micromolar concentrations, for example, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45. , 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 Inhibits expression of CD73 at concentrations of 850, 900 or 950 nM, or 1, 10 or 100 μM.

  In some embodiments, the oligonucleotides of the invention are 1, 3, 5, 9, 10, 15, 27, 30, 40, 50, 75, 82, 100, 250, 300, 500 or 740 nM or 1, 2.2. Use at a concentration of 3, 5, 6.6 or 10 μM.

  In some embodiments, the present invention refers to a pharmaceutical composition comprising an oligonucleotide of the present invention and a pharmaceutically acceptable carrier, excipient and / or diluent. In some embodiments, the pharmaceutical composition further comprises a chemotherapeutic agent, another oligonucleotide, an antibody and / or a small molecule.

  In some embodiments, the oligonucleotide or pharmaceutical composition of the invention is for use in a method of preventing and / or treating a disorder. In some embodiments, the use of an oligonucleotide or pharmaceutical composition of the invention in a method for preventing and / or treating a disorder is combined with radiation therapy. Radiation therapy can be further combined with chemotherapy (eg, platinum formulation, gemcitabine). The disorder is characterized, for example, by CD73 imbalance. That is, the level of CD73 is increased compared to the level in normal, healthy cells, tissues, organs or subjects. CD73 levels are increased, for example by increasing CD73 expression and activity, respectively. CD73 levels can be measured by any standard method, such as immunohistochemistry, Western blot, quantitative real-time PCR or QuantiGene assay known to those skilled in the art.

  The oligonucleotides or pharmaceutical compositions of the present invention can be topically or systemically, for example, oral, sublingual, nasal, subcutaneous, intravenous, intraperitoneal, intramuscular, intratumoral, intrathecal, transdermal. And / or administered rectally. Alternatively, the immune cells treated ex vivo are administered in combination or combination. Oligonucleotides may be used alone or in combination with another immunosuppressive reversion oligonucleotide of the present invention, and optionally another compound, such as another oligonucleotide, antibody, small molecule and / or chemotherapeutic agent (e.g., platinum The drug is administered in combination with Gemcitabine). In some embodiments, other oligonucleotides (i.e., not part of the invention), antibodies, and / or small molecules are used in autoimmune disorders such as autoimmune arthritis or autoimmune gastrointestinal diseases such as Inflammatory bowel disease (IBD) or colitis, immune disorders such as chronic viral infections such as immune exhaustion due to HIV infection, cardiovascular disorders, inflammatory disorders such as chronic respiratory tract inflammation, bacterial, viral and / or fungal infections such as sepsis Alternatively, it is effective in the prevention and / or treatment of Mycobacterium bovis infection, liver disorder, chronic kidney disorder, psychiatric disorder (eg schizophrenia, bipolar disorder, Alzheimer's disease) and / or cancer.

  The oligonucleotide or pharmaceutical composition of the present invention is used, for example, in a method for preventing and / or treating a solid tumor or a blood tumor. Examples of cancer that can be prevented and / or treated by the use of the oligonucleotide or pharmaceutical composition of the present invention include breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, and liver. Cancer, brain cancer, larynx, gallbladder, pancreas, testis, rectum, parathyroid gland, thyroid, adrenal gland, nerve tissue, head and neck, colon, stomach, bronchi, kidney cancer, basal cell carcinoma, squamous cell carcinoma, metastatic Skin cancer, osteosarcoma, Ewing sarcoma, reticulosarcoma, liposarcoma, myeloma, giant cell type tumor, small cell lung tumor, islet cell tumor, primary brain tumor, meningioma, acute and chronic lymphocytic and granulocytes Tumor, acute and chronic myelocytic leukemia, hairy cell tumor, adenoma, hyperplasia, medullary carcinoma, intestinal ganglion, Wilms tumor, seminoma, ovarian tumor, leiomyomater tumor, uterus Cervical dysplasia, retinoblastoma, soft tissue sarcoma Malignant carcinoid, local skin lesions, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythemia vera (polycythemia), adenocarcinoma, anaplastic astrocytoma, Glioblastoma multiforme, leukemia, or epidermoid carcinoma.

  In some embodiments, two or more oligonucleotides of the invention are administered at the same time, for example, at the same time as the pharmaceutical composition, or separately or at an interval. In other embodiments, one or more of the oligonucleotides of the present invention is another compound, e.g., another oligonucleotide (i.e. not part of the invention), antibody, small molecule and / or chemotherapy. It is administered together with the agent, for example at the same time as the pharmaceutical composition or separately or at different intervals. In some embodiments of such combinations, the immunosuppressive reversion oligonucleotide inhibits each of the expression and activity of the immunosuppressive factor, and other oligonucleotides (i.e., not part of the invention), antibodies And / or small molecules inhibit (antagonist) or stimulate (agonist) the same and / or another immunosuppressive factor and / or immunostimulatory factor. Immunosuppressive factors include, for example, IDO1, IDO2, CTLA-4, PD-1, PD-L1, LAG-3, VISTA, A2AR, CD39, CD73, STAT3, TDO2, TIM-3, TIGIT, TGF-beta, BTLA, It is selected from the group consisting of MICA, NKG2A, KIR, CD160, Chop, Xbp1 and combinations thereof. The immunostimulatory factor is selected from the group consisting of, for example, 4-1BB, Ox40, KIR, GITR, CD27, 2B4, and combinations thereof.

  An immunosuppressive factor is a factor whose expression and / or activity increases in, for example, a cell, tissue, organ or subject. An immunostimulatory factor is a factor whose level increases or decreases in a cell, tissue, organ or subject depending on the cell, tissue, organ or subject and its individual symptoms.

  The antibody combined with the oligonucleotide or pharmaceutical composition of the present invention is, for example, an anti-PD-1 antibody, an anti-PD-L1 antibody, or a bispecific antibody. Small molecules to be combined with the oligonucleotides or pharmaceutical compositions of the present invention include, for example, AMPCP (adenosine 5 ′-(α, β-methylene) diphosphate; for example, Structure 20, 2161-2173, 2012, December 5 Which acts as an ADP analog and is therefore a competitive inhibitor of CD73 activity.

  The subject of the present invention is, for example, a mammal, a bird or a fish.

The following examples illustrate various embodiments of the present invention, but the invention is not limited to these examples. The following experiments are performed on cells that endogenously express CD73. That is, the cell does not represent an artificial system containing the transfected reporter construct. Such artificial systems generally exhibit higher order inhibition and lower order IC ₅₀ values than endogenous systems that are close to therapeutically relevant in vivo systems. Furthermore, no transfection agent is used in the following experiments. That is, it performs gymnotic delivery. Transfection agents are known to increase the activity of oligonucleotides that affect IC50 values (e.g., Zhang et al., Gene Therapy, 2011, 18, 326-333; Stanton et al., Nucleic Acid Therapeutics, Vol. 22, see No. 5, 2012). Using a transfection agent as an artificial system makes it difficult or impossible to convert to a therapeutic approach, and transfection formulations have not been accepted for oligonucleotides so far. The following experiments were performed without using any transfection agent.

(Example 1)
Design of Human CD73 Antisense Oligonucleotide Design of an antisense oligonucleotide with specificity for human (h) CD73 involves the hCD73 mRNA sequence having SEQ ID NO: 1 (NM_002526.3) and SEQ ID NO: 2 (NM_0011851.4). The mCD73 mRNA sequence having was used. 14, 15, 16, and 17 mer were designed according to internal standards and neg1 (described in WO2014154843 A1) was used as a control antisense oligonucleotide in all of the experiments (Table 1). The distribution of hCD73 oligonucleotides binding to sites on hCD73 mRNA is shown in FIG.

(Example 2)
Screening efficacy of hmCD73 antisense oligonucleotides in human cancer cell lines To analyze the efficacy of the hmCD73 antisense oligonucleotides of the invention for knockdown of hmCD73 mRNA expression in cancer cell lines, FIG. 2A, FIG. 2B, FIG. 2C And as shown in FIG. 2D, a single dose of each antisense oligonucleotide (concentration: 10 μM, no transfection reagent added; this process is called gymnotic delivery) with A-172 (human glioma). Blastoma, ATCC), EFO-21 (human ovarian cystadenocarcinoma, DSMZ) and mouse 4T1 (mouse mammary cells, ATCC) cells were treated. hCD73, hGAPDH and hHPRT1 or mCD73 and mHPRT1 mRNA expression was analyzed after 3 days using QuantiGene Singleplex assay (Affymetrix). hmCD73 mRNA expression values were normalized to each of hGAPDH (A-172), hHPRT1 (EFO-21) or mHPRT1 (4T1) mRNA expression values. Notably, as shown in FIGS. 2A-2D,> 80% and> 60% knockdown efficiencies were observed for 5 (A-172 cells) antisense oligonucleotides, and> 80% knockdown Efficiency was observed for 7 (EFO-21 cells) antisense oligonucleotides, and> 90% knockdown efficiency was observed for 10 (4T1 cells) antisense oligonucleotides.

  To further analyze the effects of hmCD73 antisense oligonucleotides on CD73 mRNA expression in cancer cell lines, human SKOV-3 (human ovarian adenocarcinoma, ATCC) cells were treated with 10 μM of each antisense oligonucleotide and any transfection reagent. No treatment was added for 3 days. As a control, cells were treated with neg1, an oligonucleotide that has no sequence complementarity with any human or mouse mRNA. As a vehicle control, cells were treated with media. CD73 expression values are normalized to HPRT1 values and shown relative to untreated cells (set as 1). Significantly, hCD73 mRNA levels were reduced by ≧ 80% with two of the antisense oligonucleotides 33 tested in SKOV-3 cells (see FIG. 2E). Treatment with the control oligonucleotide neg1 did not reduce CD73 mRNA in the three cell lines.

  Accurate values of mean normalized mRNA expression of hmCD73 and the corresponding relative expression of housekeeping gene mRNA compared to untreated cells were compared to A-172 cells (Table 2 for the first and second screening rounds (Table 2 ) And Table 3 (Table 3)), EFO-21 cells (Table 4 (Table 4)), 4T1 cells (Table 5 (Table 5)) and SKOV-3 cells (Table 6 (Table 6)) are shown below. .

(Example 3)
Correlation analysis of antisense oligonucleotide potency in human EFO-21 compared to A-172 cells and human A172 compared to mouse 4T1 cells
To further select the candidates with the highest activity in the three test cell lines, A-172, EFO-21 and 4T1, correlation analysis was performed (data obtained from FIGS. 2B-2D). As shown in FIGS.3A and 3B, five powerful antisense oligonucleotides for determination of IC ₅₀ in A-172, EFO-21 and 4T1 cells, namely A05008HM (SEQ ID NO: 4), A05009HM (SEQ ID NO: 6 ), A05018HM (SEQ ID NO: 3), A05026HM (SEQ ID NO: 8) and A05028HM (SEQ ID NO: 5) (marked in black). Importantly, the control antisense oligonucleotide neg1 did not adversely affect hmCD73 expression in all three test cell lines.

(Example 4)
IC ₅₀ determination (mRNA level) of selected hmCD73 antisense oligonucleotides in SKOV-3 and EFO-21 cells, respectively
To determine the IC ₅₀ of hmCD73 antisense oligonucleotides A05008HM (SEQ ID NO: 4), A05009HM (SEQ ID NO: 6), A05018HM (SEQ ID NO: 3), A05026HM (SEQ ID NO: 8) and A05028HM (SEQ ID NO: 5) Three cells (human ovarian cancer cells, ATCC) were treated with titrated amounts of each antisense oligonucleotide (concentration: 10 μM, 3.3 μM, 1.1 μM, 370 nM, 120 nM, 41 nM, 14 nM, 4.5 nM) (FIG. 4A). hmCD73 mRNA expression was analyzed after 3 days. As shown in FIG.4A and Table 7 below, antisense oligonucleotides A05008HM (SEQ ID NO: 4), A05018HM (SEQ ID NO: 3) and A05028HM (SEQ ID NO: 5) are 86%, 88% and 75, respectively. It had the highest potency in SKOV-3 cells for down-regulation of hmCD73 mRNA compared to untreated cells with% maximum target inhibition. Concentration-dependent target knockdown in SKOV-3 cells was used to calculate the IC ₅₀ values shown in Table 7.

IC ₅₀ values of selected hmCD73-specific antisense oligonucleotides were determined in further studies of EFO-21 cells. Thus, EFO-21 cells are 10 μM, 3.3 μM, 1.1 μM, 370 nM, 120 nM, 41 nM, 14 nM, 14 nM or 4.5 nM A05018HM (SEQ ID NO: 3), A05027HM (SEQ ID NO: 30), A05028HM (SEQ ID NO: 5) and A05037HM (SEQ ID NO: 5 Treated with number 39). HCD73 mRNA expression was analyzed 3 days after treatment. FIG. 4B shows that hmCD73 mRNA expression was reduced in a concentration-dependent manner by hmCD73 antisense oligonucleotides. And IC ₅₀ values _are calculated by GraphPad Prism, shown in Table 8 (Table 8).

(Example 5)
Third screening round of hmCD73 antisense oligonucleotides in human and mouse cancer cell lines In the third screening round, new antisense oligonucleotides were designed. Such antisense oligonucleotides were based on effective antisense oligonucleotides from the first round of screening modified in length, exact location on the mRNA and chemical modification pattern. Human EFO-21 (ovarian cystadenocarcinoma) (Figure 5A), human SKOV-3 (ovarian adenocarcinoma) (Figure 5B) and mouse 4T1 (breast cancer) (Figure 5C) cells with 10 μM of each antisense oligonucleotide Treated for 3 days without adding transfection reagent. As a control, cells were treated with S6, an oligonucleotide that has no sequence complementarity with any human or mouse mRNA. As a vehicle control, cells were treated with media. Antisense oligonucleotides A05008HM, A05018HM and A05028HM that showed strong activity in the first screening round were used as a reference. Average mRNA expression of hmCD73 normalized to HPRT1 (Figure 5A-5C) compared to untreated cells (set as 1), EFO-21 cells (Table 9), SKOV-3 cells (Table 10) and 4T1 cells (Table 11) are listed. Notably, CD73 mRNA levels were ≧ 80% in 11ASO out of 13ASO tested in EFO-21 cells (see FIG. 5A) and ≧ 80% in 7ASO out of 13ASO tested in SKOV-3 cells (FIG. 5B). ), And 9ASO out of 13ASO tested in 4T1 cells was reduced by ≧ 80% (see FIG. 5C). Treatment with control oligonucleotide S6 did not strongly reduce CD73 mRNA in the three cell lines.

(Example 6)
IC ₅₀ determination (mRNA level) of selected hmCD73 antisense oligonucleotides in EFO-21 cells in the third screening round
hmCD73 antisense oligonucleotides A05038HM (SEQ ID NO: 42), A05041HM (SEQ ID NO: 45), A05042HM (SEQ ID NO: 45) and A05044HM (SEQ ID NO: 46) are potent in three cell lines EFO-21, SKOV-3 and 4T1. Single-dose activity was demonstrated. To study the concentration dependence of action and to determine IC ₅₀ values, EFO-21 cells were treated with 10 μM, 3.3 μM, 1.1 μM, 370 nM, 120 nM, 41 nM, 14 nM, or 4.5 nM of each antisense oligonucleotide. Treated. The antisense oligonucleotide A05018HM that showed strong activity in the first screening round was used as a reference. HCD73 mRNA expression was analyzed 3 days after treatment. FIG. 6 shows that hCD73 expression was reduced in a concentration-dependent manner by hmCD73 antisense oligonucleotides. IC ₅₀ values calculated by GraphPad Prism are shown in Table 12.

(Example 7)
Concentration-dependent effects on CD73 protein expression and cell viability by A05008HM (SEQ ID NO: 4), A05018HM (SEQ ID NO: 3) and A05028HM (SEQ ID NO: 5) Highly potent hmCD73 antisense oligonucleotides A05008HM (SEQ ID NO: 4), A05018HM (SEQ ID NO: 3) and A05028HM (SEQ ID NO: 5) were characterized in detail by their knockdown efficacy on hCD73 protein expression and their effect on cell viability at various concentrations. Therefore, SKOV-3 cells were treated with various concentrations of each antisense oligonucleotide for 3 days. The cells were then cultured for a further 3 days in fresh DMEM medium containing the indicated concentration of antisense oligonucleotide. Protein expression was analyzed by flow cytometry using CD73 antibody (clone AD2) and 7-AAD to examine viability. As shown in FIG. 7A, all three antisense oligonucleotides showed potent inhibition of hCD73 protein at all indicated concentrations, but treatment with neg1 had no inhibitory effect. However, cell viability was partially affected by A05028HM (FIG. 7B). In contrast, A05008HM (SEQ ID NO: 4) and A05018HM (SEQ ID NO: 3) did not affect SKOV-3 cell viability under any test conditions. Table 13 summarizes the protein knockdown efficiency of selected human CD73 antisense oligonucleotides A05008HM (SEQ ID NO: 4), A05018HM (SEQ ID NO: 3) and A05028HM (SEQ ID NO: 5) in SKOV-3 cells.

(Example 8)
Effect of CD73 protein knockdown on extracellular pyrophosphate levels in human EFO-21 and SKOV-3 cells Adenosine is one major immunosuppressive molecule produced during ATP degradation by hmCD73. Adenosine can be detected indirectly with a colorimetric phosphate assay kit (ab65622, abcam) by production of pyrophosphate during degradation of AMP to adenosine. Therefore, human SKOV-3 and EFO21 cells were treated with 5 μM antisense oligonucleotide A05018HM for 6 days (3 + 3). After 3 days, the DMEM medium was replaced with fresh DMEM medium containing 5 μM antisense oligonucleotide. After 6 days, protein knockdown of CD73 in both test cell lines was confirmed by flow cytometry (FIG. 8). As a control, cells were treated with neg1 (FIG. 8). After adding 500 μM AMP to the cells at the indicated time points, free phosphate production by EFO-21 cells (FIG. 9A) and SKOV-3 cells (FIG. 9C) in the cell supernatant was analyzed. Treatment with antisense oligonucleotide did not affect cell viability of EFO-21 cells (FIG. 9B) and SKOV-3 cells (FIG. 9D) as examined by the cell titer blue assay. Significantly, potency was clearly reduced for EFO-21 and SKOV-3 cells after treatment with A05018HM (FIGS. 9A, 9C), resulting in a comparison with cells treated with the negative control neg1. As compared with untreated cells, the phosphate concentration decreased by about 1.5 times (FIG. 9A, FIG. 9C, Table 14). Therefore, adenosine production of cancer cells can be effectively blocked by anti-CD73-specific antisense oligonucleotide treatment. Table 14 shows the phosphate concentration from the cell supernatant of SKOV-3 and EFO21 cells treated with A05018HM, and the concentration from the corresponding control reaction.

(Example 9)
Effect of hmCD73-specific antisense oligonucleotides on the degradation of extracellular AMP by human ovarian cancer cell lines and conversion to adenosine
A05018HM showed significant activity to suppress hCD73 mRNA and protein expression in human cancer cells. In the next experiment, the effect of hmCD73 antisense oligonucleotide treatment on the ability to degrade extracellular AMP into immunosuppressive adenosine was examined in EFO-21 cells.

  AMP levels as well as adenosine levels can be determined by mass spectrometry. To analyze the effect of hmCD73 antisense oligonucleotide treatment on the ability of human EFO-21 cells to convert extracellular AMP to adenosine, a total treatment time of 6 days with 5 μM antisense oligonucleotide A05018HM or control oligonucleotide S6 was used. The cells were treated for the time being. As a vehicle control, cells were treated with medium alone.

  Six days after treatment, CD73 protein expression was analyzed by flow cytometry and significantly suppressed CD73 protein expression in cells treated with A05018HM (black bar graph) compared to cells treated with S6 (white bar graph) (Figure 10A). ). To examine the ability to degrade extracellular AMP, 6 days after treatment, PBS supplemented with 300 μM AMP was replaced with cell culture medium and incubated for 6.5 hours. To study AMP degradation in the absence of cells, 300 μM AMP was added to cell-free PBS and incubated for 6.5 hours (dot bar graph).

  Subsequently, AMP levels and adenosine levels were determined by mass spectrometry in cell supernatants (cell conditioned PBS) and cell free PBS. AMP levels in the cell supernatant of untreated cells (dotted bar graph) were strongly reduced compared to AMP levels in cell-free PBS (striped bar graph) (Figure 10B), but adenosine levels increased (Figure 10B). 10C). This indicates a strong cell-related ability to convert AMP to adenosine. Significantly, AMP levels were significantly higher in the supernatant of A05018HM treated EFO-21 cells (black bar graph) when compared to the supernatant of S6 treated cells (white bar graph) or vehicle treated cells (dotted bar graph). Increased and adenosine levels decreased significantly (FIGS. 10B and 10C, Table 15 and Table 16). Thus, such data clearly shows that treatment of human EFO-21 cells with A05018HM significantly inhibits the ability of cells to convert extracellular AMP to immunosuppressive adenosine.

(Example 10)
Examination of the effect of hmCD73-specific antisense oligonucleotides on T cell proliferation in the presence or absence of extracellular AMP. The results so far of the present invention (see Example 9) show that human cancer cell lines are A05018HM Treatment has been shown to significantly inhibit their ability to convert extracellular AMP to adenosine. Since the CD39-CD73 axis plays an important role in T cell function, the effect of A05018HM on the proliferation of human CD4 ⁺ T cells in the presence or absence of extracellular AMP was examined. Thus, human CD4 ⁺ T cells are labeled with cell proliferation dye, activated with anti-CD3, and treated for 6 days with 5 μM antisense oligonucleotide A05018HM (black bar graph) or control oligonucleotide S6 (white bar graph). , Treated. In the vehicle control (striped bar graph), cells were activated with anti-CD3 alone. Subsequently, 300 μM AMP or vehicle was added to the cells on days 3 and 4 after the start of oligonucleotide treatment. On day 5 after initiation of oligonucleotide treatment, CD4 ⁺ T cells were analyzed for CD73 protein expression, proliferation, and absolute cell number using flow cytometry.

Treatment of CD4 ⁺ T cells with A05018HM strongly suppressed CD73 protein expression (FIG. 11A). In the absence of extracellular AMP, there was no difference in proliferation (FIG. 11B upper panel) or absolute cell number (FIG. 11C) between A05018HM treated cells, S6 treated cells and vehicle treated CD4 ⁺ T cells. Remarkably, supplementation with 300 μM AMP impaired the proliferation of CD4 ⁺ T cells treated with S6 or vehicle (FIG. 11B lower panel) and significantly reduced the absolute number (FIG. 11C). In contrast, proliferation of A05018HM-treated CD4 ⁺ T cells (FIG. 11B lower panel) was not impaired by supplementation of cell culture medium with AMP. Therefore, absolute T cell numbers (FIG. 11C) were hardly reduced by AMP supplementation in A05018HM treated cells.

In summary, these results revealed that supplementation of cell culture medium with AMP significantly impaired the proliferation and absolute cell number of CD73 expressing CD4 ⁺ T cells. Notably, CD73 protein knockdown by A05018HM treatment almost certainly reversed the inhibitory effect of supplemented AMP on T cell proliferation by inhibiting immunosuppressive adenosine formation.

(Example 11)
In vivo mCD73 mRNA knockdown by human / mouse cross-reactive CD73 antisense oligonucleotide (A05027HM) in mouse liver Balb / c mice after systemic administration of non-formulated oligonucleotides, selecting strong hmCD73 antisense oligonucleotide A05027HM We investigated its effect on mCD73 mRNA expression in the liver. Balb / c mice were therefore treated by subcutaneous injection of A05027HM at a dose of 20 mg / kg on days 1, 2, 3, 4, 5, 8, 10 and 12 (5 mice / group). As a control, Balb / c mice were treated with vehicle (saline, 6 mice / group). Mice were sacrificed 3 days after the last antisense oligonucleotide treatment (day 15) and livers were sampled for analysis of mCD73 mRNA levels. The results shown in FIG. 12 show mCD73 mRNA levels in the liver of A05027HM-treated or vehicle-treated mice (white bar graph). Significantly, when mice were treated with A05027HM, mCD73 mRNA levels were significantly reduced when compared to vehicle controls (p = 0.0043).

Claims (14)

  An immunosuppressive reversion oligonucleotide comprising 12 to 18 nucleotides, wherein at least one of the nucleotides is modified, and the oligonucleotide is a nucleic acid sequence and / or sequence of the extracellular enzyme (CD73) of SEQ ID NO: 1 (human) An oligonucleotide that hybridizes with the sequence of number 2 (mouse) and inhibits at least 50% of CD73 expression.   The oligonucleotide according to claim 1, wherein the modified nucleotide is selected from the group consisting of cross-linked nucleic acids, such as LNA, cET, ENA, 2'fluoro modified nucleotides, 2'O-methyl modified nucleotides and combinations thereof.   SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 42, hybridizing with CD73 of SEQ ID NO: 1 and / or SEQ ID NO: 2, comprising a sequence selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and combinations thereof The oligonucleotide according to claim 1 or 2. + G * + A * + T * T * T * C * C * C * A * G * T * G * C * + C * + A * + T (A05018HM),
+ G * + C * + T * G * T * G * C * A * C * G * T * C * + G * + T * + T (A05008HM),
+ T * + G * + A * T * T * T * C * C * C * A * G * T * G * C * + C * + A * + T (A05028HM),
+ G * + G * + C * T * G * T * G * C * A * C * G * T * C * + G * + T (A05009HM),
+ C * + T * + C * A * G * A * A * T * T * G * G * A * A * + A * + T * + T (A05019HM),
+ A * + C * + T * C * G * A * C * A * C * T * T * G * G * + T * + G * + C (A05026HM),
+ C * + T * + G * T * G * C * A * C * G * T * C * + G * + T * + T (A05001HM),
+ A * + G * C * A * C * G * T * T * G * G * G * T * + T * + C (A05002HM),
+ A * + C * + G * G * T * G * A * A * C * C * A * + G * + A * + T (A05003HM),
+ G * + C * + A * T * A * G * G * C * C * T * G * + G * + A * + C (A05004H),
+ C * + T * + C * G * A * C * A * C * T * T * G * G * + T * + G (A050005HM),
+ A * + C * + T * C * G * A * C * A * C * T * T * + G * + G * + T (A05006HM),
+ G * + G * + C * A * C * T * C * G * A * C * A * + C * + T * + T (A05007HM),
+ G * + T * + C * C * T * C * C * C * A * C * C * A * + C * + G * + A (A05010HM),
+ G * + T * C * C * T * C * C * C * A * C * C * A * C * G * + A (A05011HM),
+ T * + G * T * C * C * T * C * C * C * A * C * C * + A * + C * + G (A05012HM),
+ G * + A * G * T * G * T * C * C * T * C * C * C * A * C * + C (A05013HM),
+ T * + C * + G * A * C * A * C * T * T * G * G * T * + G * + C * + A (A05014HM),
+ C * + T * + C * G * A * C * A * C * T * T * G * G * + T * + G * + C (A05015HM),
+ A * + C * + T * C * G * A * C * A * C * T * T * + G * G * T * + G (A05016HM),
+ C * + A * + C * T * C * G * A * C * A * C * T * T * + G * + G * + T (A05017HM),
+ T * + G * + T * C * C * T * C * C * C * A * C * C * A * + C * + G * + A (A05020HM),
+ G * + T * G * T * C * C * T * C * C * C * A * C * C * + A * + C * + G (A05021HM),
+ G * + A * G * T * G * T * C * C * T * C * C * C * A * C * + C * + A (A05022HM),
+ G * T * + G * T * T * G * G * A * G * T * G * T * C * C * + T * + C (A05023HM),
+ C * + T * + C * G * A * C * A * C * T * T * G * G * T * + G * + C * + A (A05024HM),
+ C * + T * + C * G * A * C * A * C * T * T * G * G * T * G * + C * + A (A05025HM),
+ G * + C * + A * C * T * C * G * A * C * A * C * T * T * + G * + G * + T (A05027HM),
+ G * + T * + G * T * C * C * T * C * C * C * A * C * C * A * + C * + G * + A (A05029HM),
+ A * + G * + T * G * T * C * C * T * C * C * C * A * C * C * + A * + C * + G (A05030HM),
+ G * + A * G * T * G * T * C * C * T * C * C * C * A * C * C * A * + C (A05031HM),
+ G * A * G * T * G * T * C * C * T * C * C * C * A * C * + C * A * + C (A05032HM),
+ G * A * G * T * G * T * C * C * T * C * C * C * A * C * C * + A * + C (A05033HM),
+ A * + C * + T * C * G * A * C * A * C * T * T * G * G * T * + G * + C * + A (A05034HM),
+ A * + C * + T * C * G * A * C * A * C * T * T * G * G * T * G * + C * + A (A05035HM),
+ G * + G * + C * A * C * T * C * G * A * C * A * C * T * T * + G * + G * + T (A05036HM),
+ G * + G * CA * C * T * C * G * A * C * A * C * T * T * + G * + G * + T (A05037HM),
+ G * + C * T * G * T * G * C * A * C * G * T * C * + G * + T * + T (A05038HM),
+ C * G * + G * C * T * G * T * G * C * A * C * G * T * C * G * + T * + T (A05039HM),
+ G * + G * + C * T * G * T * G * C * A * C * G * T * C * + G * + T (A05040HM),
+ G * + A * + T * T * T * C * C * C * A * G * T * G * C * C * + A * + T (A05041HM),
+ G * + A * T * T * T * C * C * C * A * G * T * G * C * + C * + A * + T (A05042HM),
+ T * + G * + A * T * T * T * C * C * C * A * G * T * G * + C * C * + A * + T (A05043HM),
+ T * + G * + A * T * T * T * C * C * C * A * G * T * G * C * C * + A * + T (A05044HM),
+ C * + A * + T * G * A * T * T * T * C * C * C * A * G * T * G * + C * + C (A05045HM),
And selected from the group consisting of these,
The oligonucleotide according to any one of claims 1 to 3, wherein + represents an LNA nucleotide and * represents an internucleotide phosphorothioate (PTO) bond.   The oligonucleotide according to any one of claims 1 to 4, which inhibits the expression of CD73 at a nanomolar concentration.   6. A pharmaceutical composition comprising the immunosuppressive reverting oligonucleotide according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier, excipient and / or diluent.   7. The pharmaceutical composition according to claim 6, further comprising a chemotherapeutic agent, such as a platinum preparation, gemcitabine, another oligonucleotide, an antibody and / or a small molecule.   8. The pharmaceutical composition according to claim 7, wherein the other oligonucleotide, antibody and / or small molecule inhibits or stimulates an immunosuppressive factor and / or an immunostimulatory factor.   Immunosuppressive factors are IDO1, IDO2, CTLA-4, PD-1, PD-L1, LAG-3, VISTA, A2AR, CD39, CD73, STAT3, TDO2, TIM-3, TIGIT, TGF-beta, BTLA, MICA, 9. The pharmaceutical composition according to claim 8, selected from the group consisting of NKG2A, KIR, CD160, Chop, Xbp1 and combinations thereof.   9. The pharmaceutical composition according to claim 8, wherein the immunostimulatory factor is selected from the group consisting of 4-1BB, Ox40, KIR, GITR, CD27, 2B4 and combinations thereof.   The immunosuppressive reverting oligonucleotide according to any one of claims 1 to 5 or any one of claims 6 to 10 for use in a method of preventing and / or treating a disorder involving an imbalance of CD73. The pharmaceutical composition according to item.   12. The immunosuppressive reverting oligonucleotide or pharmaceutical composition for use according to claim 11, wherein the disorder is an autoimmune disorder, an immune disorder, a mental disorder and / or a cancer.   Cancer is breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer, larynx, gallbladder, pancreas, testis, rectum, parathyroid gland, thyroid gland, adrenal gland , Nerve tissue, head and neck, colon, stomach, bronchi, kidney cancer, basal cell carcinoma, squamous cell carcinoma, metastatic skin cancer, osteosarcoma, Ewing sarcoma, reticulosarcoma, liposarcoma, myeloma, giant cell type Tumor, small cell lung tumor, islet cell tumor, primary brain tumor, meningioma, acute and chronic lymphocytic and granulocytic tumor, acute and chronic myelocytic leukemia, hairy cell tumor, adenoma, hyperplasia, medullary Cancer, intestinal ganglion, Wilms tumor, seminoma, ovarian tumor, leiomyoma, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, local skin lesion, rhabdomyosarcoma, Kaposi sarcoma, Osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, true red Sphere vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforme, leukemias, or epidermoid carcinoma, immunosuppression return oligonucleotide or pharmaceutical composition for use according to claim 11.   14. An immunosuppressive reverting oligonucleotide or pharmaceutical composition for use according to any one of claims 11 to 13, suitable for local or systemic administration.