技术领域technical field
本公开涉及与胞外酶(NT5E或CD73)的核酸序列杂交的免疫抑制恢复寡核苷酸，以及包含这种免疫抑制恢复寡核苷酸和药用载体、赋形剂和/或稀释剂的药物组合物。The present disclosure relates to an immunosuppressive recovery oligonucleotide hybridized to a nucleic acid sequence of an extracellular enzyme (NT5E or CD73), and an immunosuppressive recovery oligonucleotide comprising such an immunosuppressive recovery oligonucleotide and a pharmaceutically acceptable carrier, excipient and/or diluent pharmaceutical composition.
背景技术Background technique
近年来，通过应用免疫疗法，特别是通过所谓的“免疫检查点”的抑制剂，对如恶性肿瘤的几种不同疾病的治疗非常成功。这些检查点是免疫系统中的分子，其上调(共刺激分子)或下调信号。治疗方法的概念基于内源性抗肿瘤免疫反应的激活。例如，许多癌症分别通过抑制T细胞和NK细胞活性来保护自己免受免疫系统的侵害。免疫检查点调节剂，即刺激剂或抑制剂，例如针对CTLA-4、PD-1、PD-L1、LAG-3、VISTA、A2AR、BTLA、IDO、CD39、CD73、STAT3、TDO2、TIM-3、MICA、NKG2A、KIR、TIGIT、TGF-β、Ox40、GITR、CD27、CD160、2B4和4-1Bb中的一种或多种。Several different diseases such as malignancies have been treated with great success in recent years by the application of immunotherapy, in particular by inhibitors of so-called "immune checkpoints". These checkpoints are molecules in the immune system that upregulate (co-stimulatory molecules) or downregulate signals. The concept of the therapeutic approach is based on the activation of the endogenous antitumor immune response. For example, many cancers protect themselves from the immune system by suppressing T-cell and NK-cell activity, respectively. Immune checkpoint modulators, i.e. stimulators or inhibitors, e.g. against CTLA-4, PD-1, PD-L1, LAG-3, VISTA, A2AR, BTLA, IDO, CD39, CD73, STAT3, TDO2, TIM-3 One or more of , MICA, NKG2A, KIR, TIGIT, TGF-β, Ox40, GITR, CD27, CD160, 2B4 and 4-1Bb.
CD73需要被认为是提高对不同类型癌症的免疫力的一种新颖且有前景的候选物。CD73是一种胞外酶(NTPdase)，并且催化AMP向免疫抑制性腺苷的转化。CD73协同作用，但在CD39的下游，已知将ATP转化为ADP，并将ADP转化为AMP。腺苷通过腺苷受体A1、腺苷受体A2A、腺苷受体A2B和腺苷受体A3发挥其作用。表达腺苷受体并因此可能受腺苷的免疫调节作用影响的免疫细胞的范围包括T淋巴细胞、自然杀伤(NK)细胞、NKT细胞、巨噬细胞、DC、中性粒细胞、肥大细胞和B细胞。CD73 needs to be considered as a novel and promising candidate for boosting immunity against different types of cancer. CD73 is an extracellular enzyme (NTPdase) and catalyzes the conversion of AMP to immunosuppressive adenosine. CD73 acts synergistically but downstream of CD39 and is known to convert ATP to ADP and ADP to AMP.Adenosine exerts its effects through adenosine receptor A1, adenosine receptorA2A , adenosine receptorA2B and adenosine receptor A3. The range of immune cells that express adenosine receptors and thus may be affected by the immunomodulatory effects of adenosine include T lymphocytes, natural killer (NK) cells, NKT cells, macrophages, DCs, neutrophils, mast cells and B cells.
CD73存在于大多数组织和许多细胞类型中，包括淋巴细胞的亚群、巨噬细胞、树突细胞、内皮细胞和上皮细胞。缺氧诱导小鼠微血管内皮细胞中的CD73 mRNA、蛋白质表达并增加CD73活性。特别地，CD73在许多不同的人(实体和血液)肿瘤中高度表达，并且其升高的表达和活性与肿瘤侵袭性和转移相关并且具有较短的患者存活期。CD73的RNA表达和酶活性在不同的乳腺癌细胞系中是可变的。CD73 is present in most tissues and in many cell types, including subsets of lymphocytes, macrophages, dendritic cells, endothelial cells, and epithelial cells. Hypoxia induces CD73 mRNA, protein expression and increases CD73 activity in mouse microvascular endothelial cells. In particular, CD73 is highly expressed in many different human (solid and hematological) tumors, and its elevated expression and activity correlates with tumor aggressiveness and metastasis and with shorter patient survival. CD73 RNA expression and enzymatic activity are variable in different breast cancer cell lines.
将死的癌细胞将ATP释放到肿瘤微环境中的细胞外空间。活的肿瘤细胞通常表达高水平的CD39和CD73，并将ATP转化为免疫抑制性腺苷。由此，肿瘤细胞能够进行不受控制的增殖和扩增。通过与淋巴细胞上的A2A或A2B受体结合，腺苷介导对这些细胞的免疫抑制信号。例如，T细胞的增殖、细胞毒性细胞因子产生和激活受到抑制。NK细胞显示出降低的细胞毒性潜力。腺苷诱导巨噬细胞中的替代激活(免疫抑制性M2表型)，导致促炎性细胞因子产生减少，但免疫抑制性细胞因子IL-10的产生增加。CD73作为相关治疗靶标在不同肿瘤中的重要作用由以下事实强调：使用CD73或A2A受体敲除小鼠的肿瘤模型显示出改善的疾病结果。Dying cancer cells release ATP into the extracellular space in the tumor microenvironment. Viable tumor cells often express high levels of CD39 and CD73 and convert ATP to immunosuppressive adenosine. As a result, tumor cells are capable of uncontrolled proliferation and expansion. By binding toA2A orA2B receptors on lymphocytes, adenosine mediates immunosuppressive signals on these cells. For example, T cell proliferation, cytotoxic cytokine production and activation are inhibited. NK cells display reduced cytotoxic potential. Adenosine induces alternative activation in macrophages (immunosuppressive M2 phenotype), resulting in decreased production of pro-inflammatory cytokines but increased production of the immunosuppressive cytokine IL-10. The important role of CD73 as a relevant therapeutic target in different tumors is underscored by the fact that tumor models using CD73 or A2Areceptor knockout mice show improved disease outcomes.
抗人CD73单克隆抗体如Innate Pharma的抗CD73抗体(例如，Innate PharmaPoster#iph_poster_aarc2016_cd73)目前正在基于免疫肿瘤学细胞的试验中进行临床前研究。然而，由于空间位阻，针对CD73的单克隆抗体可能无法定位于肿瘤微环境中。此外，CD73的不可水解的小分子抑制剂如AMPCP(腺苷5'-(α,β-亚甲基)二磷酸；例如，结构20,2161-2173,2012年12月5日)(其为ADP类似物)竞争性地抑制CD73活性，已经在动物模型中体外和体内测试，但需要相对高的浓度和重复给药以成功阻断CD73酶活性。Anti-human CD73 monoclonal antibodies such as Innate Pharma's anti-CD73 antibody (eg, Innate PharmaPoster #iph_poster_aarc2016_cd73) are currently undergoing preclinical studies in immuno-oncology cell-based assays. However, monoclonal antibodies against CD73 may fail to localize in the tumor microenvironment due to steric hindrance. In addition, nonhydrolyzable small molecule inhibitors of CD73 such as AMPCP (adenosine 5'-(α,β-methylene) diphosphate; e.g., Structure 20, 2161-2173, Dec. 5, 2012) (which is ADP analogs) competitively inhibit CD73 activity and have been tested in vitro and in vivo in animal models, but require relatively high concentrations and repeated dosing to successfully block CD73 enzymatic activity.
免疫疗法已经导致长期缓解，但到目前为止只对小患者群体。原因可能是许多免疫检查点和可选的其它免疫抑制机制参与例如免疫系统和肿瘤细胞之间的相互作用。免疫检查点和潜在的其它机制的组合可以根据肿瘤和受试者的个体状况而不同，以逃避身体的防御。Immunotherapy has resulted in long-term remission, but so far only in small patient groups. The reason may be that many immune checkpoints and optionally other immunosuppressive mechanisms are involved in, for example, the interaction between the immune system and tumor cells. The combination of immune checkpoints and potentially other mechanisms can vary according to the tumor and the individual condition of the subject to evade the body's defenses.
为了抑制几种免疫抑制机制，使用抗体和/或小分子的常用方法不适合或几乎不适合，因为分子靶标位于细胞内或不具有酶活性。因此，安全有效地抑制如CD73的“免疫检查点”功能的药剂将是治疗患有例如由该酶活性影响的疾病或病症的患者的重要补充。To inhibit several immunosuppressive mechanisms, common approaches using antibodies and/or small molecules are not or barely suitable because the molecular targets are intracellular or not enzymatically active. Therefore, an agent that safely and effectively inhibits the function of an "immune checkpoint" such as CD73 would be an important addition to the treatment of patients suffering from diseases or conditions affected, for example, by the activity of this enzyme.
本发明的寡核苷酸分别在抑制CD73的表达和活性方面非常成功。寡核苷酸的作用方式不同于抗体或小分子的作用方式，并且寡核苷酸对于例如以下是非常有利的The oligonucleotides of the invention were very successful in inhibiting the expression and activity of CD73, respectively. The mode of action of oligonucleotides is different from that of antibodies or small molecules, and oligonucleotides are very advantageous for, for example,
(i)肿瘤组织在实体肿瘤中的渗透，(i) penetration of tumor tissue in solid tumors,
(ii)分别阻断靶标的多项功能和活动，(ii) block multiple functions and activities of the target separately,
(iii)寡核苷酸彼此或与抗体或小分子的组合，和(iii) oligonucleotides in combination with each other or with antibodies or small molecules, and
(iv)抑制对于抗体是不可接近的或通过小分子可抑制的细胞内效果。(iv) Inhibition of intracellular effects that are inaccessible to antibodies or inhibitable by small molecules.
因此，通过反义寡核苷酸在mRNA水平上靶向癌症和免疫细胞上的CD73表达是开发和改进例如分别针对不同癌症和免疫疾病的免疫疗法的有前景的最新技术方法。Therefore, targeting CD73 expression on cancer and immune cells at the mRNA level by antisense oligonucleotides is a promising state-of-the-art approach to develop and improve e.g. immunotherapies against different cancers and immune diseases, respectively.
发明内容Contents of the invention
本发明涉及寡核苷酸，如包含约10至20个核苷酸的免疫抑制恢复(immunosuppression-reverting)寡核苷酸，其中至少一个核苷酸被修饰。寡核苷酸与例如SEQ ID NO.1(人)的胞外酶CD73的核酸序列和/或SEQ ID NO.2(小鼠)的序列杂交。修饰的核苷酸例如选自由桥接核酸(例如，LNA、cET、ENA、2’氟修饰的核苷酸或2'O-甲基修饰的核苷酸)组成的组。在一些实施方式中，寡核苷酸抑制至少50％的CD73表达，并且在一些实施方式中，寡核苷酸以纳摩尔浓度抑制CD73的表达。The present invention relates to oligonucleotides, such as immunosuppression-reverting oligonucleotides comprising about 10 to 20 nucleotides, wherein at least one nucleotide is modified. The oligonucleotide hybridizes to, for example, 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). Modified nucleotides are for example selected from the group consisting of bridging nucleic acids (e.g. LNA, cET, ENA, 2'fluoro-modified nucleotides or 2'O-methyl-modified nucleotides). In some embodiments, the oligonucleotide inhibits expression of CD73 by at least 50%, and in some embodiments, the oligonucleotide inhibits expression of CD73 at nanomolar concentrations.
与RNAi相比，反义寡核苷酸具有显着的优势。可以在体外转染反义寡核苷酸而不转染试剂，因此与使用专门转染RNAi的转染试剂的转染相比，该转染更接近体内条件。在不同组织中体内全身给予反义寡核苷酸是可能的，而体内给予RNAi依赖于递送系统，如GalNAc，例如在肝脏中。此外，反义寡核苷酸比RNAi短，因此在合成和吸收入细胞方面较不复杂。RNAi规律地显示同样可以启动RNAi的随从链的脱靶(off-target)效果。随从链RISC加载是RNAi药物的重要问题，因为随从链可能将RNAi活性导向非预期的靶标，导致毒副作用。“(参见Chackalamannil,Rotella,Ward,Comprehensive Modicinal Chemistry IIIElsevier,03.06.2017)。反义寡核苷酸不包含随从链。Antisense oligonucleotides have significant advantages over RNAi. Antisense oligonucleotides can be transfected in vitro without transfection reagents, thus bringing the transfection closer to in vivo conditions than transfection using transfection reagents specifically designed to transfect RNAi. Systemic administration of antisense oligonucleotides in different tissues is possible in vivo, whereas in vivo administration of RNAi relies on delivery systems such as GalNAc, for example in the liver. Furthermore, antisense oligonucleotides are shorter than RNAi and thus less complex to synthesize and take up into cells. RNAi regularly exhibits off-target effects that can also initiate the RNAi's follower chain. Entourage strand RISC loading is an important issue for RNAi drugs because the follower strand may direct RNAi activity to unintended targets, leading to toxic side effects. "(see Chackalamannil, Rotella, Ward, Comprehensive Modicinal Chemistry III Elsevier, 03.06.2017). Antisense oligonucleotides do not contain a follower strand.
本发明还涉及药物组合物，其包含本发明的免疫抑制恢复寡核苷酸和可选的药用载体、赋形剂和/或稀释剂。在一些实施方式中，该药物组合物另外包含化学治疗剂，如铂或吉西他滨，另一种寡核苷酸、抗体和/或例如在肿瘤治疗中有效的小分子。The present invention also relates to pharmaceutical compositions comprising the immunosuppressive restoration oligonucleotides of the present invention and optionally pharmaceutically acceptable carriers, excipients and/or diluents. In some embodiments, the pharmaceutical composition additionally comprises a chemotherapeutic agent, such as platinum or gemcitabine, another oligonucleotide, an antibody and/or a small molecule that is effective, for example, in tumor therapy.
在一些实施方式中，本发明的寡核苷酸与另一种寡核苷酸、抗体和/或小分子组合，这些化合物中的每一种在药物组合物中分离或组合，其中寡核苷酸、抗体和/或小分子抑制或刺激免疫抑制因子，如IDO1、IDO2、CTLA-4、PD-1、PD-L1、LAG-3、VISTA、A2AR,CD39、CD73、STAT3,TDO2、TIM-3、TIGIT、TGF-β、BTLA、MICA、NKG2A、KIR、CD160、Chop和/或Xbp1。另外或可替代地，寡核苷酸、抗体和/或小分子抑制或刺激免疫刺激因子，例如4-1BB、Ox40、KIR、GITR、CD27和/或2B4。In some embodiments, an oligonucleotide of the invention is combined with another oligonucleotide, antibody and/or small molecule, each of these compounds is isolated or combined in a pharmaceutical composition, wherein the oligonucleotide Acids, antibodies and/or small molecules inhibit or stimulate immunosuppressive factors such as IDO1, IDO2, CTLA-4, PD-1, PD-L1, LAG-3, VISTA, A2AR, CD39, CD73, STAT3, TDO2, TIM- 3. TIGIT, TGF-β, BTLA, MICA, NKG2A, KIR, CD160, Chop and/or Xbp1. Additionally or alternatively, the oligonucleotides, antibodies and/or small molecules inhibit or stimulate immunostimulatory factors such as 4-1BB, Ox40, KIR, GITR, CD27 and/or 2B4.
此外，本发明涉及本发明的寡核苷酸或药物组合物在预防和/或治疗其中涉及CD73失衡的疾病的方法中的用途。在一些实施方式中，该病症是例如自身免疫病症，例如自身免疫性关节炎或胃肠道自身免疫疾病，例如炎性肠病(IBD)或结肠炎、免疫病症，例如由于慢性病毒感染如HIV感染引起的免疫衰竭、心血管疾病、炎性疾病，例如慢性气道炎症、细菌、病毒和/或真菌感染，例如败血症或牛结核分支杆菌(Mycobacterium bovis)感染、肝脏疾病、慢性肾病、精神疾病和/或癌症CD73具有许多生理学作用，如调节屏障功能、适应低氧、缺血预处理、抗炎、白细胞渗出。CD73在癌细胞上的表达和激活与不良的预后相关并且可能促进转移。CD73促进人乳腺癌细胞的粘附、迁移、侵袭和胶质瘤细胞的增值，并且这些过程与酶的腺苷生产无关。在一些实施方式中，例如局部或全身给予本发明的寡核苷酸或药物组合物。Furthermore, the invention relates to the use of an oligonucleotide or a pharmaceutical composition according to the invention in a method of preventing and/or treating a disease in which an imbalance of CD73 is involved. In some embodiments, the disorder is, for example, an autoimmune disorder, such as autoimmune arthritis or an autoimmune disease of the gastrointestinal tract, such as inflammatory bowel disease (IBD) or colitis, an immune disorder, such as due to chronic viral infection such as HIV Immune failure due to infection, cardiovascular disease, inflammatory disease such as chronic airway inflammation, bacterial, viral and/or fungal infection such as sepsis or Mycobacterium bovis infection, liver disease, chronic kidney disease, psychiatric disorders And/or cancer CD73 has many physiological roles, such as regulation of barrier function, adaptation to hypoxia, ischemic preconditioning, anti-inflammation, leukocyte extravasation. CD73 expression and activation on cancer cells correlates with poor prognosis and may promote metastasis. CD73 promotes adhesion, migration, and invasion of human breast cancer cells and proliferation of glioma cells, and these processes are independent of enzymatic adenosine production. In some embodiments, for example, the oligonucleotides or pharmaceutical compositions of the invention are administered locally or systemically.
本文引用或参考的所有文件(“本文引用的文件”)，以及本文引用的文件中引用或参考的所有文件，以及本文或本文通过引证并入的任何文件提及的任何产品的任何制造商的说明、描述、产品规格和产品说明书，并且可以用于本发明的实践中。更具体地，所有参考文件通过引用并入，其程度如同每个单独的文件被具体和单独地指出通过引证并入。All documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, and any manufacturer of any product mentioned herein or in any document incorporated by reference herein Instructions, descriptions, product specifications and product instructions, and may be used in the practice of the present invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
附图说明Description of drawings
图1描绘了人(h)CD73反义寡核苷酸结合位点在SEQ ID No.1的hCD73 mRNA(NM_002526.3)上的分布以及它们的(一个或多个)修饰和长度。将hCD73反义寡核苷酸与SEQ IDNo.1的hCD73 mRNA序列比对。不同的灰度表示不同的LNA修饰，且符号表示反义寡核苷酸的不同长度。Figure 1 depicts the distribution of human (h)CD73 antisense oligonucleotide binding sites on hCD73 mRNA of SEQ ID No. 1 (NM_002526.3) and their modification(s) and length. Align the hCD73 antisense oligonucleotide with the hCD73 mRNA sequence of SEQ ID No.1. Different gray shades indicate different LNA modifications, and symbols indicate different lengths of antisense oligonucleotides.
图2A-2E描绘了在第一轮和第二轮筛选(图2A和2B(第1部分和第2部分))中在人癌细胞系A-172(人胶质母细胞瘤)中和在一轮筛选中(图2C(第1部分和第2部分))中在EFO-21(人卵巢囊腺癌)中、在一轮筛选中在一个鼠细胞系4T1(乳腺癌)中(图2D(第1部分和第2部分))和在一轮筛选中在人类SKOV-3(人类卵巢腺癌)细胞中(图2E)的hmCD73反义寡核苷酸的hCD73mRNA敲除功效。用10μM的相应的反义寡核苷酸处理A-172、EFO-21、4T1和SKOV-3细胞3天。作为对照，用neg1处理细胞，neg1是具有序列CGTTTAGGCTATGTACTT的反义寡核苷酸(描述于WO2014154843 A1中)。描绘了相对于未处理的细胞的残留的人或小鼠CD73 mRNA表达。将表达值归一化为管家基因GAPDH或HPRT1的表达值。描绘的是一式三份的孔的平均值+/-SD。Figure 2A-2E depicts in the first and second rounds of screening (Figure 2A and 2B (part 1 and part 2)) in the human cancer cell line A-172 (human glioblastoma) and in In one round of screening (Figure 2C (part 1 and part 2)) in EFO-21 (human ovarian cystadenocarcinoma), in one round of screening in a murine cell line 4T1 (breast cancer) (Figure 2D (Parts 1 and 2)) and hCD73 mRNA knockdown efficacy of hmCD73 antisense oligonucleotides in human SKOV-3 (human ovarian adenocarcinoma) cells (Fig. 2E) in one round of screening. A-172, EFO-21, 4T1 and SKOV-3 cells were treated with 10 μM of the corresponding antisense oligonucleotides for 3 days. As a control, cells were treated with neg1 , an antisense oligonucleotide with the sequence CGTTTAGGCTATGTACTT (described in WO2014154843 A1 ). Residual human or mouse CD73 mRNA expression relative to untreated cells is depicted. Expression values were normalized to those of the housekeeping genes GAPDH or HPRT1. Depicted are mean values +/- SD of triplicate wells.
图3A和3B显示了人EFO-21细胞与人A-172细胞相比(图3A)和人A-172细胞与小鼠4T1细胞相比(图3B)的hmCD73反义寡核苷酸功效的相关性分析。Figures 3A and 3B show a graph of hmCD73 antisense oligonucleotide efficacy in human EFO-21 cells compared to human A-172 cells (Figure 3A) and human A-172 cells compared to mouse 4T1 cells (Figure 3B). correlation analysis.
图4A和4B显示了SKOV-3细胞和EFO-21(人卵巢癌)中选择的hmCD73反义寡核苷酸的浓度依赖性hm CD73mRNA敲除，选择的hmCD73反义寡核苷酸为A05008HM(SEQ ID No.4)、A05009HM(SEQ ID No.6)、A05018HM(SEQ ID No.3)、A05026HM(SEQ ID No.8)和A05028HM(SEQ ID No.5)(图4A)和EFO 21细胞中的A05018HM(SEQ ID No.3)、A05027HM(SEQ IDNo.30)、A05028HM(SEQ ID No.5)和A05037HM(SEQ ID No.39)(图4B)。SKOV-3和EFO-21细胞用指定浓度的相应反义寡核苷酸处理3天。描绘了与未处理的对照细胞(设定为100)相比的残留的hCD73表达。将hCD73 mRNA表达值归一化为管家基因HPRT1的表达。描述的是一式三份的孔的平均值+/-SD。浓度依赖性靶标敲除用于计算表7的SKOV-3细胞和表8的EFO-21细胞的IC50值。Figures 4A and 4B show the concentration-dependent knockdown of hmCD73 mRNA in SKOV-3 cells and EFO-21 (human ovarian carcinoma) by the selected hmCD73 antisense oligonucleotide, the selected hmCD73 antisense oligonucleotide is 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) (Figure 4A) and EFO 21 cells A05018HM (SEQ ID No. 3), A05027HM (SEQ ID No. 30), A05028HM (SEQ ID No. 5) and A05037HM (SEQ ID No. 39) in (FIG. 4B). SKOV-3 and EFO-21 cells were treated with the corresponding antisense oligonucleotides at the indicated concentrations for 3 days. Residual hCD73 expression compared to untreated control cells (set as 100) is depicted. hCD73 mRNA expression values were normalized to the expression of the housekeeping gene HPRT1. Depicted are mean values +/- SD of triplicate wells. Concentration-dependent target knockouts were used to calculateIC50 values for SKOV-3 cells in Table 7 and EFO-21 cells in Table 8.
图5A至5C描绘了hmCD73反义寡核苷酸在人和小鼠癌细胞系中的hmCD73 mRNA敲除功效。EFO-21(人卵巢囊腺癌(图5A))、SKOV-3(人卵巢腺癌(图5B))和4T1(小鼠乳腺癌(图5C))细胞用10μM的相应的反义寡核苷酸处理3天。描绘了相对于未处理细胞(设定为1)的残留hmCD73 mRNA表达。将表达值归一化为管家基因HPRT1的表达。描绘的是一式三份的孔的平均值+/-SD。Figures 5A to 5C depict hmCD73 mRNA knockdown efficacy of hmCD73 antisense oligonucleotides in human and mouse cancer cell lines. EFO-21 (human ovarian cystadenocarcinoma (Figure 5A)), SKOV-3 (human ovarian adenocarcinoma (Figure 5B)) and 4T1 (mouse breast cancer (Figure 5C)) cells were treated with 10 μM of the corresponding antisense oligo Glycolic acid treatment for 3 days. Residual hmCD73 mRNA expression relative to untreated cells (set to 1) is depicted. Expression values were normalized to the expression of the housekeeping gene HPRT1. Depicted are mean values +/- SD of triplicate wells.
图6显示了用指定浓度的相应的反义寡核苷酸处理EFO-21细胞3天。将hCD73 mRNA表达值归一化为管家基因HPRT1的表达。描绘了相对于未处理细胞(设定为100)的残留hCD73 mRNA表达。描绘的是一式三份的孔的平均值+/-SD。Figure 6 shows the treatment of EFO-21 cells with the corresponding antisense oligonucleotides at the indicated concentrations for 3 days. hCD73 mRNA expression values were normalized to the expression of the housekeeping gene HPRT1. Residual hCD73 mRNA expression relative to untreated cells (set as 100) is depicted. Depicted are mean values +/- SD of triplicate wells.
图7A和7B描绘了通过A05008HM(SEQ ID No.4)、A05018HM(SEQ ID No.3)和A05028HM(SEQ ID No.5)的浓度依赖性hCD73 mRNA和蛋白质敲除。通过流式细胞术分析CD73的蛋白质表达和细胞活力(图7A)，并在用指定的反义寡核苷酸处理6天后，进行SKOV-3细胞的7-AAD染色(图7B)。作为对照，用neg1类似地处理细胞。描绘了相对于未用任何反义寡核苷酸处理的对照细胞的数量(＝1)的CD73蛋白表达的中值荧光强度(图7A)和总死亡细胞(7-AAD阳性细胞)(图7B)。Figures 7A and 7B depict concentration-dependent knockdown of hCD73 mRNA and protein by A05008HM (SEQ ID No. 4), A05018HM (SEQ ID No. 3) and A05028HM (SEQ ID No. 5). CD73 protein expression and cell viability were analyzed by flow cytometry ( FIG. 7A ), and 7-AAD staining of SKOV-3 cells was performed after 6 days of treatment with the indicated antisense oligonucleotides ( FIG. 7B ). As a control, cells were similarly treated with Neg1. Median fluorescence intensity of CD73 protein expression (Fig. 7A) and total dead cells (7-AAD positive cells) relative to the number of control cells not treated with any antisense oligonucleotide (=1) are depicted (Fig. ).
图8显示了反义寡核苷酸处理后SKOV3和EFO-21细胞上CD73蛋白表达的敲除。在用A05018HM(SEQ ID No.3)处理6天后分析两种测试的细胞系上的CD73蛋白表达。作为对照，用neg1类似地处理细胞。描绘了相对于未用任何反义寡核苷酸处理的对照细胞的表达(＝1)的CD73蛋白表达的中值荧光强度。Figure 8 shows the knockdown of CD73 protein expression on SKOV3 and EFO-21 cells after antisense oligonucleotide treatment. CD73 protein expression on the two tested cell lines was analyzed after 6 days of treatment with A05018HM (SEQ ID No. 3). As a control, cells were similarly treated with Neg1. The median fluorescence intensity of CD73 protein expression is depicted relative to the expression (=1) of control cells not treated with any antisense oligonucleotide.
图9A-9D描绘了hCD73敲除对SKOV-3和EFO-21细胞的细胞外焦磷酸水平和细胞活力的影响。来自用A05018HM(SEQ ID No.3)处理6天的EFO-21细胞(图9A)和SKOV-3细胞(图9C)的细胞上清液中分析作为腺苷产生的间接量度的焦磷酸酯的生成。在测量之前，将外源AMP在指定时间点以500μM添加到细胞中。使用细胞滴度蓝(cell titer blue)试验测试使用A05018HM(SEQ ID No.3)的处理对EFO-21细胞(图9B)和SKOV-3细胞(图9D)的细胞活力的影响。Figures 9A-9D depict the effect of hCD73 knockdown on extracellular pyrophosphate levels and cell viability in SKOV-3 and EFO-21 cells. Analysis of pyrophosphate as an indirect measure of adenosine production in cell supernatants from EFO-21 cells (Fig. 9A) and SKOV-3 cells (Fig. 9C) treated with A05018HM (SEQ ID No. 3) for 6 days generate. Exogenous AMP was added to cells at 500 μM at indicated time points prior to measurement. The effect of treatment with A05018HM (SEQ ID No. 3) on the cell viability of EFO-21 cells ( FIG. 9B ) and SKOV-3 cells ( FIG. 9D ) was tested using the cell titer blue assay.
图10A至10C显示以5μM用CD73特异性反义寡核苷酸A05018HM(黑色柱)或对照寡核苷酸S6(白色柱)处理的EFO-21细胞，总处理时间为6天。通过流式细胞术分析hCD73蛋白表达。图10A描绘了相对于未处理细胞(斑点柱；设定为1)的残留hCD73表达。为了分析降解细胞外AMP的能力，在终止前6.5小时将300μM的AMP加入细胞或无细胞PBS(条带柱)中。图10B显示相对于不含AMP补充PBS的细胞(设定为1)的相对AMP水平，且图10C显示细胞上清液(细胞条件的PBS)中的绝对腺苷浓度。描绘的是对于无细胞PBS的条件的3孔的平均值+/-SD或单个值。Figures 10A to 10C show EFO-21 cells treated with CD73-specific antisense oligonucleotide A05018HM (black bars) or control oligonucleotide S6 (white bars) at 5 μM for a total treatment time of 6 days. hCD73 protein expression was analyzed by flow cytometry. Figure 10A depicts residual hCD73 expression relative to untreated cells (dotted columns; set to 1). To analyze the ability to degrade extracellular AMP, 300 [mu]M AMP was added to cells or cell-free PBS (strip column) 6.5 hours before termination. Figure 10B shows relative AMP levels relative to cells supplemented with PBS without AMP (set as 1), and Figure 10C shows absolute adenosine concentrations in cell supernatants (cell-conditioned PBS). Depicted are the mean +/- SD or individual values of 3 wells for the condition of cell-free PBS.
图11A至11C描绘了用细胞增殖染料标记，用抗CD3激活并用5μM的CD73特异性反义寡核苷酸A05018HM(黑色柱)或对照寡核苷酸S6(白色柱)处理5天的总处理时间的人CD4+T细胞。在载体对照(条纹柱)中，仅用抗CD3激活细胞。随后，在寡核苷酸处理开始后第3天和第4天将300μM的AMP或载体加入细胞中。图11A显示CD73蛋白表达，图11B描绘了增殖，且图11C显示CD4+T细胞的绝对细胞数，其在寡核苷酸处理开始后第5天使用流式细胞术分析。描绘的是一式三份的孔的平均值+/-SD。Figures 11A to 11C depict total treatment labeled with cell proliferation dye, activated with anti-CD3 and treated with 5 μM of CD73-specific antisense oligonucleotide A05018HM (black bars) or control oligonucleotide S6 (white bars) for 5 days time human CD4+ T cells. In vehicle controls (striped columns), cells were activated with anti-CD3 only. Subsequently, 300 μM of AMP or vehicle was added to the cells on days 3 and 4 after the initiation of oligonucleotide treatment. Figure 11A shows CD73 protein expression, Figure 11B depicts proliferation, and Figure 11C shows absolute cell numbers of CD4+ T cells analyzed using flow cytometry at day 5 after initiation of oligonucleotide treatment. Depicted are mean values +/- SD of triplicate wells.
图12描绘了hmCD73反义寡核苷酸(A05027HM)处理对小鼠肝脏中mCD73mRNA表达的体内作用。图12中描绘的结果显示A05027HM或媒介物处理的小鼠(白色柱)的肝脏中的mCD73 mRNA水平。Figure 12 depicts the in vivo effect of hmCD73 antisense oligonucleotide (A05027HM) treatment on mCD73 mRNA expression in mouse liver. Results depicted in Figure 12 show mCD73 mRNA levels in livers of A05027HM or vehicle treated mice (white bars).
图13显示了SEQ ID No.1(NM_002526.3)的hCD73 mRNA。Figure 13 shows hCD73 mRNA of SEQ ID No. 1 (NM_002526.3).
具体实施方式Detailed ways
本发明首次提供了人和鼠寡核苷酸，其与外核苷酶CD73的mRNA序列杂交并分别抑制例如肿瘤细胞或肿瘤相关免疫细胞上的CD73的表达和活性。结果，ATP水平增加，且其降解产物如ADP、AMP和免疫抑制性腺苷水平降低。所有这些效果分别导致抗肿瘤免疫细胞增加，免疫激活(例如，通过细胞毒性T细胞或NK细胞)和肿瘤细胞的识别和消除。因此，本发明的寡核苷酸代表了用于预防和/或治疗其中CD73表达和活性分别增加的疾病的方法的感兴趣且高效的工具。The present invention provides for the first time human and murine oligonucleotides that hybridize to the mRNA sequence of the exonucleosidase CD73 and inhibit, for example, the expression and activity of CD73 on tumor cells or tumor-associated immune cells, respectively. As a result, ATP levels are increased and levels of its degradation products such as ADP, AMP, and immunosuppressive adenosine are decreased. All of these effects lead to an increase in antitumor immune cells, immune activation (for example, by cytotoxic T cells or NK cells) and recognition and elimination of tumor cells, respectively. Thus, the oligonucleotides of the invention represent interesting and efficient tools for methods of prevention and/or treatment of diseases in which CD73 expression and activity, respectively, are increased.
在下文中，将更详细地描述本发明的元素。这些元素与特定实施方式一起列出，然而，应该理解，它们可以以任何方式和任何数量组合以产生另外的实施方式。不应将各种描述的实施例和实施方式解释为将本发明仅限于明确描述的实施方式。应该理解，该描述支持和包含将明确描述的实施方式与任何数量的公开元素组合的实施方式。此外，除非上下文另有说明，否则本申请中的所有描述的元素的任何排列和组合应该被认为是由本申请的说明书公开。Hereinafter, elements of the present invention will be described in more detail. These elements are listed with particular embodiments, however, it should be understood that they may be combined in any way and in any number to create additional embodiments. The various described examples and implementations should not be construed to limit the invention to only the expressly described implementations. It should be understood that this description supports and encompasses combinations of the explicitly described embodiments with any number of disclosed elements. Furthermore, unless the context dictates otherwise, any permutation and combination of all described elements in this application should be considered to be disclosed by the specification of this application.
在整个说明书和权利要求书中，除非上下文另有要求，否则词语“包括(comprise)”或如“包括(comprises)”或“包括(comprising)”的变体将被理解为暗示包括所述成员、整数或步骤，或成员、整数或步骤的组，但不排除任何其它成员、整数或步骤，或成员、整数或步骤的组。除非在本文中另有说明或上下文明显矛盾，否则在描述本发明的上下文中使用的术语“一个”和“一种”和“该”以及类似的指代(特别是在权利要求的上下文中)应被解释为涵盖单数和复数。本文中对数值范围的描述仅旨在用作单独提及落入该范围内的每个单独值的速记法。除非本文另有说明，否则每个单独的值被并入说明书中，如同其在本文中单独引用一样。除非本文另有说明或上下文明显矛盾，否则本文所述的所有方法均可以任何合适的顺序进行。本文提供的任何和所有实例或示例性语言(例如，“如”，“例如”)的使用仅旨在更好地说明本发明，而不是对另外要求保护的本发明的范围构成限制。说明书中的任何语言都不应被解释为表示对于本发明的实践必不可少的任何未要求保护的要素。Throughout the specification and claims, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of said members , integer or step, or group of members, integers or steps, without excluding any other member, integer or step, or group of members, integers or steps. Unless otherwise indicated herein or clearly contradicted by context, the terms "a" and "an" and "the" and similar designations when used in the context of describing the invention (especially in the context of the claims) shall be construed to cover both the singular and the plural. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited 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 language (eg, "such as," "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
本发明的寡核苷酸是例如反义寡核苷酸，其包含10至25个核苷酸、10至15个核苷酸、15至20个核苷酸、12至18个核苷酸或14至17个核苷酸或由其组成。例如，寡核苷酸包含10、11、12、13、14、15、16、17、18、19、20或25个核苷酸或由其组成。本发明的寡核苷酸包含至少一个修饰的核苷酸。修饰的核苷酸是例如，桥连核苷酸如锁核酸(LNA，例如，2’,4’-LNA)、cET、ENA、2'氟修饰的核苷酸、2'O-甲基修饰的核苷酸或它们的组合。在一些实施方式中，本发明的寡核苷酸包含具有相同或不同修饰的核苷酸。在一些实施方式中，本发明的寡核苷酸包含修饰的磷酸主链，其中磷酸是例如硫代磷酸或甲基磷酸或两者。Oligonucleotides of the invention are, for example, antisense oligonucleotides comprising 10 to 25 nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 12 to 18 nucleotides or or consisting of 14 to 17 nucleotides. For example, an oligonucleotide comprises or consists of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 nucleotides. The oligonucleotides of the invention comprise at least one modified nucleotide. Modified nucleotides are, for example, bridging nucleotides such as locked nucleic acids (LNA, e.g., 2',4'-LNA), cET, ENA, 2'fluoro-modified nucleotides, 2'O-methyl-modified nucleotides or combinations thereof. In some embodiments, 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, where the phosphate is, for example, phosphorothioate or methylphosphate or both.
本发明的寡核苷酸在寡核苷酸的3'-末端和/或5'-末端和/或寡核苷酸内的任何位置包含一个或多个修饰的核苷酸，其中修饰的核苷酸在1、2、3、4、5或6个修饰的核苷酸的行中，或修饰的核苷酸与一个或多个未修饰的核苷酸组合。下表1给出了包含修饰的核苷酸的寡核苷酸的本实施方式，例如由(+)表示的LNA和由(*)表示的硫代磷酸(PTO)。分别包含表1的序列由其组成的寡核苷酸可以包含任何其它修饰的核苷酸以及修饰的和未修饰的核苷酸的任何其它组合。表1的寡核苷酸与人和鼠CD73的mRNA杂交：The oligonucleotides of the present invention comprise one or more modified nucleotides at the 3'-end and/or 5'-end of the oligonucleotide and/or anywhere within the oligonucleotide, wherein the modified core The nucleotides are in rows of 1, 2, 3, 4, 5 or 6 modified nucleotides, or modified nucleotides are combined with one or more unmodified nucleotides. Table 1 below presents this embodiment of oligonucleotides comprising modified nucleotides, such as LNA indicated by (+) and phosphorothioate (PTO) indicated by (*). The oligonucleotides each comprising the sequence of Table 1 consist of may comprise any other modified nucleotides and any other combination of modified and unmodified nucleotides. The oligonucleotides of Table 1 hybridize to human and mouse CD73 mRNA:
表1：与例如分别为SEQ ID No.1和SEQ ID No.2的人和鼠CD73杂交的反义寡核苷酸的列表。Neg1是代表未与SEQ ID No.1和SEQ ID No.2的CD73杂交的阴性对照的反义寡核苷酸。Table 1: List of antisense oligonucleotides that hybridize to human and murine CD73 eg SEQ ID No. 1 and SEQ ID No. 2, respectively. Neg1 is an antisense oligonucleotide representing a negative control that does not hybridize to CD73 of SEQ ID No. 1 and SEQ ID No. 2.
本发明的寡核苷酸例如与SEQ ID No.1和/或SEQ ID No.2的人或鼠CD73的mRNA杂交。这种寡核苷酸称为CD73反义寡核苷酸。The oligonucleotides of the invention hybridize, for example, to human or murine CD73 mRNA of SEQ ID No. 1 and/or SEQ ID No. 2. Such oligonucleotides are referred to as CD73 antisense oligonucleotides.
在一些实施方式中，本发明的寡核苷酸抑制至少约50％、55％、60％、65％、70％、75％、80％、85％、90％、92％、94％、95％、96％、97％、98％、99％或100％的CD73，例如人、鼠CD73表达。因此，本发明的寡核苷酸是免疫抑制恢复寡核苷酸，其例如在细胞、组织、器官或受试者中恢复免疫抑制。本发明的寡核苷酸以纳摩尔或微摩尔浓度，例如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、850、900或950nM，或1、10或100μM的浓度抑制CD73的表达。In some embodiments, oligonucleotides of the invention inhibit at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95% %, 96%, 97%, 98%, 99% or 100% of CD73, eg human, mouse CD73 expression. Thus, the oligonucleotides of the invention are immunosuppression restoration oligonucleotides, which restore immunosuppression, for example, in a cell, tissue, organ or subject. The oligonucleotides of the present invention are available in nanomolar or micromolar concentrations, such as 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, Concentrations of 800, 850, 900 or 950 nM, or 1, 10 or 100 μM inhibited the expression of CD73.
在一些实施方式中，本发明的寡核苷酸以1、3、5、9、10、15、27、30、40、50、75、82、100、250、300、500或740nM，或1、2.2、3、5、6.6或10μM的浓度使用。In some embodiments, an oligonucleotide of the invention is administered at 1, 3, 5, 9, 10, 15, 27, 30, 40, 50, 75, 82, 100, 250, 300, 500 or 740 nM, or 1 , 2.2, 3, 5, 6.6 or 10 μM concentrations were used.
在一些实施方式中，本发明涉及药物组合物，其包含本发明的寡核苷酸和药用载体、赋形剂和/或稀释剂。在一些实施方式中，药物组合物还包含化学治疗剂、另一种寡核苷酸、抗体和/或小分子。In some embodiments, the present invention relates 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, antibody and/or small molecule.
在一些实施方式中，本发明的寡核苷酸或药物组合物用于预防和/或治疗病症的方法。在一些实施方式中，本发明的寡核苷酸或药物组合物用于预防和/或治疗病症的方法与放射疗法结合使用。放射疗法可以进一步与化学疗法(例如铂、吉西他滨)结合使用。该病症特征在于例如CD73失衡，即与正常健康细胞、组织、器官或受试者中的水平相比，CD73水平增加。CD73水平分别通过例如增加的CD73表达和活性而增加。CD73水平可以通过任何标准方法测量，例如本领域技术人员已知的免疫组织化学、蛋白质印迹、定量实时PCR或QuantiGene测定。In some embodiments, an oligonucleotide or pharmaceutical composition of the invention is used in a method of preventing and/or treating a disorder. In some embodiments, methods of using oligonucleotides or pharmaceutical compositions of the invention for the prevention and/or treatment of disorders are used in conjunction with radiation therapy. Radiation therapy can further be combined with chemotherapy (eg platinum, gemcitabine). The condition is characterized by, for example, a CD73 imbalance, ie an increased level of CD73 compared to the level in a normal healthy cell, tissue, organ or subject. CD73 levels are increased by, for example, increased 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 assays known to those skilled in the art.
本发明的寡核苷酸或药物组合物局部或全身给予，例如口腔、舌下、经鼻、皮下、静脉内、腹膜内、肌肉内、瘤内、鞘内、透皮和/或直肠给予。可替换地或组合地，给予离体处理的免疫细胞。寡核苷酸单独给予或与本发明的另一种免疫抑制恢复寡核苷酸组合给予，并可选地与另一种化合物如另一种寡核苷酸、抗体、小分子和/或化学治疗剂(例如铂、吉西他滨)组合给予。在一些实施方式中，其他的寡核苷酸(即，不是本发明的一部分)、抗体和/或小分子有效预防和/或治疗自身免疫性疾病，例如自身免疫性关节炎或胃肠道自身免疫疾病，如炎性肠病(IBD)或结肠炎、免疫疾病，例如由于慢性病毒感染如HIV感染引起的免疫衰竭、心血管疾病、炎性疾病例如慢性气道炎症、细菌、病毒和/或真菌感染，例如败血症或牛结核分支杆菌感染、肝脏疾病、慢性肾病、精神病症(例如，精神分裂症、双相情感障碍、阿尔茨海默病)和/或癌症。The oligonucleotides or pharmaceutical compositions of the invention are administered locally or systemically, eg, orally, sublingually, nasally, subcutaneously, intravenously, intraperitoneally, intramuscularly, intratumorally, intrathecally, transdermally and/or rectally. Alternatively or in combination, ex vivo treated immune cells are administered. The oligonucleotide is administered alone or in combination with another immunosuppressive restoration oligonucleotide of the invention, and optionally with another compound such as another oligonucleotide, antibody, small molecule and/or chemical Therapeutic agents (eg platinum, gemcitabine) are given in combination. In some embodiments, other oligonucleotides (i.e., not part of the invention), antibodies and/or small molecules are effective in preventing and/or treating autoimmune diseases, such as autoimmune arthritis or the gastrointestinal tract itself Immune disorders such as inflammatory bowel disease (IBD) or colitis, immune disorders such as immune failure due to chronic viral infection such as HIV infection, cardiovascular disease, inflammatory diseases such as chronic airway inflammation, bacterial, viral and/or Fungal infections, such as sepsis or Mycobacterium bovis infection, liver disease, chronic kidney disease, psychiatric disorders (eg, schizophrenia, bipolar disorder, Alzheimer's disease), and/or cancer.
本发明的寡核苷酸或药物组合物用于例如预防和/或治疗实体瘤或血液肿瘤的方法。通过使用本发明的寡核苷酸或药物组合物可预防和/或可治疗的癌症的实例是乳腺癌、肺癌、恶性黑色素瘤、淋巴瘤、皮肤癌、骨癌、前列腺癌、肝癌、脑癌、喉癌、胆囊癌、胰腺癌、睾丸癌、直肠癌、甲状旁腺癌、甲状腺癌、肾上腺癌、神经组织癌、头颈癌、结肠癌、胃癌、支气管癌、肾癌、基底细胞癌、鳞状细胞癌、转移性皮肤癌、骨肉瘤、尤文氏肉瘤、网状细胞肉瘤、脂肪肉瘤、骨髓瘤、巨细胞瘤、小细胞肺肿瘤、胰岛细胞瘤、原发性脑肿瘤、脑膜瘤、急性和慢性淋巴细胞和粒细胞瘤、急性和慢性髓性白血病、毛细胞瘤、腺瘤、增生、髓样癌、肠神经节瘤、肾母细胞瘤(Wilm’s tumor)、精原细胞瘤、卵巢肿瘤、平滑肌瘤、宫颈非典型增生(dysplasia)、视网膜母细胞瘤、软组织肉瘤、恶性类癌、局部皮损、横纹肌肉瘤、卡波西肉瘤、骨原性肉瘤、恶性高钙血症、肾细胞瘤、真性红细胞增多症、腺癌、间变性星形细胞瘤、多形性胶质母细胞瘤、白血病或表皮样癌。The oligonucleotides or pharmaceutical compositions of the invention are used, for example, in methods of preventing and/or treating solid tumors or hematological tumors. Examples of cancers that are preventable and/or treatable by using the oligonucleotides or pharmaceutical compositions of the present invention are breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer , laryngeal cancer, gallbladder cancer, pancreatic cancer, testicular cancer, rectal cancer, parathyroid cancer, thyroid cancer, adrenal gland cancer, nerve tissue cancer, head and neck cancer, colon cancer, gastric cancer, bronchial cancer, kidney cancer, basal cell carcinoma, squamous cell carcinoma Cell carcinoma, metastatic skin cancer, osteosarcoma, Ewing's sarcoma, reticulum cell sarcoma, liposarcoma, myeloma, giant cell tumor, small cell lung tumor, islet cell tumor, primary brain tumor, meningioma, acute and chronic lymphocytic and granulocytoma, acute and chronic myelogenous leukemia, pilocytoma, adenoma, hyperplasia, medullary carcinoma, enteric ganglioma, Wilm's tumor, seminoma, ovarian tumor , leiomyoma, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, localized skin lesions, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell polycythemia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforme, leukemia, or epidermoid carcinoma.
在一些实施方式中，本发明的两种或更多种寡核苷酸在同一时间点一起给予，例如在药物组合物中，或单独给予，或以交错的间隔给予。在其它实施方式中，本发明的一种或多种寡核苷酸与另一种化合物，如另一种寡核苷酸(即，不是本发明的一部分)、抗体、小分子和/或化学治疗剂，在同一时间点，例如在药物组合物中一起给予，或单独给予或以交错的间隔给予。在这些组合的一些实施方式中，免疫抑制恢复寡核苷酸分别抑制免疫抑制因子和其它寡核苷酸(即，不是本发明的一部分)，抗体和/或其小分子抑制物(拮抗剂)或刺激物(激动剂)和/或另一种免疫抑制因子和/或免疫刺激因子。免疫抑制因子选自由例如IDO1、IDO2、CTLA-4、PD-1、PD-L1、LAG-3、VISTA、A2AR、CD39、CD73、STAT3,TDO2、TIM-3、TIGIT、TGF-β、BTLA、MICA、NKG2A、KIR、CD160、Chop、Xbp1和它们的组合组成的组。免疫刺激因子选自由例如4-1BB、Ox40、KIR、GITR、CD27、2B4和它们的组合组成的组。In some embodiments, two or more oligonucleotides of the invention are administered together at the same point in time, eg, in a pharmaceutical composition, or administered separately, or at staggered intervals. In other embodiments, one or more oligonucleotides of the invention are combined with another compound, such as another oligonucleotide (i.e., not part of the invention), antibody, small molecule and/or chemical The therapeutic agents are administered together at the same point in time, eg, in a pharmaceutical composition, either separately or at staggered intervals. In some embodiments of these combinations, the immunosuppressive restoring oligonucleotides inhibit immunosuppressive factors and other oligonucleotides (i.e., not part of the invention), antibodies and/or small molecule inhibitors (antagonists) thereof, respectively or a stimulant (agonist) and/or another immunosuppressive and/or immunostimulatory factor. The immunosuppressive factor is selected from, for example, IDO1, IDO2, CTLA-4, PD-1, PD-L1, LAG-3, VISTA, A2AR, CD39, CD73, STAT3, TDO2, TIM-3, TIGIT, TGF-β, BTLA, The panel consisting of MICA, NKG2A, KIR, CD160, Chop, Xbp1 and combinations thereof. The immunostimulatory factor is selected from the group consisting of eg 4-1BB, Ox40, KIR, GITR, CD27, 2B4 and combinations thereof.
免疫抑制因子是其表达和/或活性例如在细胞、组织、器官或受试者中增加的因子。免疫刺激因子是其水平取决于细胞、组织、器官或受试者及其个体状况而在细胞、组织、器官或受试者中升高或降低的因子。An immunosuppressive factor is a factor whose expression and/or activity is increased, eg, in a cell, tissue, organ or subject. An immunostimulatory factor is a factor whose level is increased or decreased in a cell, tissue, organ or subject depending on the cell, tissue, organ or subject and its individual condition.
与本发明的寡核苷酸或药物组合物组合的抗体是例如抗PD-1抗体、抗PD-L1抗体或双特异性抗体。与本发明的寡核苷酸或药物组合物组合的小分子是例如AMPCP(腺苷5′-(α,β-亚甲基)二磷酸酯，；例如Structure20，2161-2173,December 5,2012)，其作用为ADP类似物并且因此是CD73活性的竞争性抑制剂。Antibodies combined with oligonucleotides or pharmaceutical compositions of the invention are eg anti-PD-1 antibodies, anti-PD-L1 antibodies or bispecific antibodies. Small molecules combined with oligonucleotides or pharmaceutical compositions of the invention are for example AMPCP (adenosine 5'-(α,β-methylene) diphosphate; for example Structure20, 2161-2173, December 5, 2012 ), which acts as an ADP analog and is thus a competitive inhibitor of CD73 activity.
本发明的受试者是例如哺乳动物、鸟或鱼。A subject of the invention is eg a mammal, a bird or a fish.
实施例Example
以下实施例说明了本发明的不同实施方式，但本发明不限于这些实施例。对内源表达CD73的细胞(即该细胞不代表包含转染的报道基因构建体的人工系统)进行以下实验。与更接近治疗相关的体内系统的内源系统相比，这种人工系统通常显示出更高程度的抑制和更低的IC50值。此外，在以下实验中，不使用转染剂，即进行自主递送(gymnoticdelivery)。已知转染剂会增加影响IC 50值的寡核苷酸的活性(参见例如Zhang et,al,Gene Therapy,2011,18,326-333；Stanton et,al,Nucleic Acid Therapeutics,Vol.22,No.5,2012)。由于使用转染剂的人工系统难以或不可能转化为治疗方法，并且目前尚无批准用于寡核苷酸的转染制剂，因此在没有任何转染剂的情况下进行以下实验。The following examples illustrate different embodiments of the invention, but the invention is not limited to these examples. The following experiments were performed on cells endogenously expressing CD73 (ie, the cells did not represent an artificial system comprising a transfected reporter construct). Such artificial systems generally show higher degrees of inhibition and lowerIC50 values than endogenous systems that more closely resemble therapeutically relevant in vivo systems. Furthermore, in the following experiments, gymnotic delivery was performed without using a transfection agent. Transfection agents are known to increase the activity of oligonucleotides that affect IC50 values (see e.g. Zhang et, al, Gene Therapy, 2011, 18, 326-333; Stanton et, al, Nucleic Acid Therapeutics, Vol.22, No. 5, 2012). Since artificial systems using transfection agents are difficult or impossible to translate into therapeutics, and there are currently no transfection formulations approved for oligonucleotides, the following experiments were performed without any transfection agents.
实施例1：人CD73反义寡核苷酸的设计Embodiment 1: the design of human CD73 antisense oligonucleotide
为了设计对人(h)CD73具有特异性的反义寡核苷酸，使用具有SEQ ID No.1的hCD73 mRNA序列(NM_002526.3)和具有SEQ ID No.2的mCD73mRNA序列(NM_0011851.4)。根据内部标准设计14、15、16和17聚体，在所有实验中使用neg1(在WO2014154843 A1中描述)作为对照反义寡核苷酸(表1)。hCD73 mRNA上的hCD73寡核苷酸结合位点的分布如图1所示。For the design of antisense oligonucleotides specific to human (h)CD73, the hCD73 mRNA sequence (NM_002526.3) with SEQ ID No. 1 and the mCD73 mRNA sequence (NM_0011851.4) with SEQ ID No.2 were used . 14, 15, 16 and 17mers were designed according to internal standards, neg1 (described in WO2014154843 A1 ) was used as control antisense oligonucleotide in all experiments (Table 1). The distribution of hCD73 oligonucleotide binding sites on hCD73 mRNA is shown in Figure 1 .
实施例2：hmCD73反义寡核苷酸在人癌细胞系中的功效筛选Example 2: Efficacy screening of hmCD73 antisense oligonucleotides in human cancer cell lines
为了分析本发明的hCD39反义寡核苷酸在癌细胞系中对hmCD73 mRNA表达的敲除的功效，使用单剂量(浓度：10μM，不添加任何转染试剂，该过程称为自主递送)的相应的反义寡核苷酸处理A-172(人胶质母细胞瘤，ATCC)、EFO-21(人卵巢囊腺癌，DSMZ)和小鼠4T1(小鼠乳腺细胞，ATCC)细胞；如图2A、2B、2C和2D所示。使用QuantiGene Singleplex测定(Affymetrix)在三天后分析hCD73、hGAPDH和hHPRT1或mCD73和mHPRT1mRNA表达。hmCD73mRNA表达值分别归一化至hGAPDH(A-172)、hHPRT1(EFO-21)或mHPRT1(4T1)mRNA表达值。引人注目的是，如图2A至2D所示，观察到对于5种(A-172细胞)反义寡核苷酸分别>80％和>60％的敲除效率；对于7种(EFO-21细胞反义寡核苷酸)>80％的敲除效率，对于10种(4T1细胞)反义寡核苷酸>90％的敲除效率。In order to analyze the efficacy of the hCD39 antisense oligonucleotides of the present invention in knocking out hmCD73 mRNA expression in cancer cell lines, a single dose (concentration: 10 μM, without adding any transfection reagent, this process is called autonomous delivery) Corresponding antisense oligonucleotides treated A-172 (human glioblastoma, ATCC), EFO-21 (human ovarian cystadenocarcinoma, DSMZ) and mouse 4T1 (mouse mammary gland cells, ATCC) cells; as Figures 2A, 2B, 2C and 2D. hCD73, hGAPDH and hHPRT1 or mCD73 and mHPRT1 mRNA expression were analyzed after three days using the QuantiGene Singleplex assay (Affymetrix). hmCD73 mRNA expression values were normalized to hGAPDH(A-172), hHPRT1(EFO-21) or mHPRT1(4T1) mRNA expression values, respectively. Strikingly, as shown in Figures 2A to 2D, knockout efficiencies of >80% and >60% were observed for 5 (A-172 cells) antisense oligonucleotides; 21 cell antisense oligonucleotides)>80% knockout efficiency, for 10 kinds (4T1 cells) antisense oligonucleotides>90% knockout efficiency.
为了进一步分析hmCD73反义寡核苷酸对癌细胞系中CD73 mRNA表达的影响，将人SKOV-3(人卵巢腺癌，ATCC)细胞用10μM的相应的反义寡核苷酸处理3天，不添加任何转染试剂。作为对照，用neg1处理细胞，neg1是一种与任何人或小鼠mRNA没有序列互补性的寡核苷酸。作为载体对照，用培养基处理细胞。将CD73表达值归一化至HPRT1值，并相对于未处理的细胞(设定为1)显示。引人注目的是，SKOV-3细胞中的33种测试的反义寡核苷酸中的2种的hCD73 mRNA水平降低了≥80％(参见图2E)。用对照寡核苷酸neg 1处理不会降低这三种细胞系中的CD73 mRNA。To further analyze the effect 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 the corresponding antisense oligonucleotides for 3 days, No transfection reagent was added. As a control, cells were treated with Neg1, an oligonucleotide with no sequence complementarity to any human or mouse mRNA. As a vehicle control, cells were treated with medium. CD73 expression values were normalized to HPRT1 values and displayed relative to untreated cells (set to 1). Strikingly, 2 out of 33 tested antisense oligonucleotides reduced hCD73 mRNA levels by > 80% in SKOV-3 cells (see Figure 2E). Treatment with the control oligonucleotide Neg 1 did not decrease CD73 mRNA in these three cell lines.
与未处理的细胞相比，hmCD73的平均归一化的mRNA表达的确切值和管家基因mRNA的相应的相对表达在下面对于A-172细胞(表2和3为第1和第2轮筛选)、EFO-21细胞(表4)、4T1细胞(表5)和SKOV-3细胞(表6)给出：The exact values of mean normalized mRNA expression of hmCD73 and the corresponding relative expression of housekeeping gene mRNA compared to untreated cells are below for A-172 cells (Tables 2 and 3 for round 1 and round 2 screening) , EFO-21 cells (Table 4), 4T1 cells (Table 5) and SKOV-3 cells (Table 6) give:
表2：第一轮筛选的与未处理的细胞相比的反义寡核苷酸处理的A-172细胞中的平均归一化hCD73 mRNA表达值的列表。Table 2: List of mean normalized hCD73 mRNA expression values in antisense oligonucleotide-treated A-172 cells compared to untreated cells for the first round of screening.
表3：第二轮筛选的与未处理的细胞相比的反义寡核苷酸处理的A-172细胞中的平均归一化hCD73 mRNA表达值的列表。Table 3: List of mean normalized hCD73 mRNA expression values in antisense oligonucleotide-treated A-172 cells compared to untreated cells for the second round of screening.
表4：与未处理的细胞相比，反义寡核苷酸处理的EFO-21细胞中的平均归一化hCD73 mRNA表达值的列表。Table 4: List of mean normalized hCD73 mRNA expression values in antisense oligonucleotide-treated EFO-21 cells compared to untreated cells.
表5：与未处理的细胞相比，反义寡核苷酸处理的4T1细胞中的平均归一化mCD73mRNA表达值的列表。Table 5: List of mean normalized mCD73 mRNA expression values in antisense oligonucleotide-treated 4T1 cells compared to untreated cells.
表6：相对于未处理的细胞(设定为1)，反义寡核苷酸处理的SKOV-3细胞中的平均归一化hmCD73 mRNA表达值。Table 6: Mean normalized hmCD73 mRNA expression values in antisense oligonucleotide-treated SKOV-3 cells relative to untreated cells (set as 1).
实施例3：，人EFO-21与人A-172细胞相比以及人A172与小鼠4T1细胞相比的反义寡核苷酸功效的相关性分析。Example 3: Correlation analysis of antisense oligonucleotide efficacy of human EFO-21 compared with human A-172 cells and human A172 compared with mouse 4T1 cells.
为了进一步在三种测试的细胞系A-172、EFO-21和4T1中选择具有最高活性的候选物，进行相关性分析(来自图2B至2D的数据)。如图3A和图3B所示，选择5种有效的反义寡核苷酸，即A05008HM(SEQ ID No.4)、A05009HM(SEQ ID No.6)、A05018HM(SEQ ID No.3)、A05026HM(SEQ ID No.8)和A05028HM(SEQ ID No.5)(标记为黑色)用于测定A-172、EFO-21和4T1细胞中的IC50。重要的是，对照反义寡核苷酸neg1对所有三种测试的细胞系中的hCD39的表达没有负面影响。To further select the candidate with the highest activity among the three tested cell lines A-172, EFO-21 and 4T1, a correlation analysis was performed (data from Figures 2B to 2D). As shown in Figure 3A and Figure 3B, select 5 effective antisense oligonucleotides, 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) were used to determineIC50 in A-172, EFO-21 and 4T1 cells. Importantly, the control antisense oligonucleotide neg1 had no negative effect on hCD39 expression in all three cell lines tested.
实施例4：分别在SKOV-3细胞和EFO-21细胞中选择的hmCD73反义寡核苷酸的IC50测定(mRNA水平)Example 4: IC50 determination (mRNA level) of hmCD73 antisense oligonucleotides selected in SKOV-3 cells and EFO-21 cells respectively
为了确定hmCD73反义寡核苷酸A05008HM(SEQ ID No.4)、A05009HM(SEQ IDNo.6)、A05018HM(SEQ ID No.3)、A05026HM(SEQ ID No.8)和A05028HM(SEQ ID No.5)的IC50，用滴定量的相应的反义寡核苷酸(浓度：10μM、3.3μM、1.1μM、370nM、120nM、41nM、14nM或4.5nM)处理SKOV-3细胞(人卵巢癌细胞，ATCC)(图4A)。三天后分析hmCD73 mRNA表达。如图4A和下表7所示，与未处理的细胞相比，反义寡核苷酸A05008HM(SEQ ID No.4)、A05018HM(SEQ ID No.3)和A05028HM(SEQ ID No.5)在hCD39 mRNA的下调方面在SKOV-3细胞中具有最高的效力，最大靶标抑制率分别为86％、88％和75％。SKOV-3细胞中的浓度依赖性靶标敲除用于计算表7中所示的IC50值：In order to determine 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) IC50 , treated SKOV-3 cells (human ovarian cancer cells , ATCC) (Fig. 4A). hmCD73 mRNA expression was analyzed three days later. As shown in Figure 4A and Table 7 below, compared with untreated cells, antisense oligonucleotides A05008HM (SEQ ID No.4), A05018HM (SEQ ID No.3) and A05028HM (SEQ ID No.5) It had the highest potency in the downregulation of hCD39 mRNA in SKOV-3 cells with maximal target inhibition of 86%, 88% and 75%, respectively. Concentration-dependent target knockdown in SKOV-3 cells was used to calculate theIC50 values shown in Table 7:
表7：SKOV-3细胞中选择的hmCD73反义寡核苷酸的IC50值和靶标抑制的概览。Table 7: Overview ofIC50 values and target inhibition of selected hmCD73 antisense oligonucleotides in SKOV-3 cells.
在EFO-21细胞中的进一步测试中测定选择的hmCD73特异性反义寡核苷酸的IC50值。因此，用10μM、3.3μM、1.1μM、370nM、120nM、41nM、14nM或4.5nM的A05018HM(SEQ IDNo.3)、A05027HM(SEQ ID No.30)、A05028HM(SEQ ID No.5)和A05037HM(SEQ ID No.39)处理EFO-21细胞。在处理3天后分析hCD73 mRNA表达。图4B描绘了通过hmCD73反义寡核苷酸的hmCD73 mRNA表达的浓度依赖性降低。通过GraphPad Prism计算IC50值，并显示在表8中：IC50 values of selected hmCD73-specific antisense oligonucleotides were determined in further tests in EFO-21 cells. Therefore, A05018HM (SEQ ID No. 3), A05027HM (SEQ ID No. 30), A05028HM (SEQ ID No. 5) and A05037HM ( SEQ ID No.39) to treat EFO-21 cells. hCD73 mRNA expression was analyzed 3 days after treatment. Figure 4B depicts concentration-dependent reduction of hmCD73 mRNA expression by hmCD73 antisense oligonucleotides.IC50 values were calculated by GraphPad Prism and shown in Table 8:
表8：在EFO-21细胞中选择的hmCD73反义寡核苷酸的IC50值和靶标抑制Table 8:IC50 values and target inhibition of selected hmCD73 antisense oligonucleotides in EFO-21 cells
实施例5：hmCD73反义寡核苷酸在人和小鼠癌细胞系中的第三轮筛选Example 5: The third round of screening of hmCD73 antisense oligonucleotides in human and mouse cancer cell lines
对于第三轮筛选，设计了新的反义寡核苷酸。这些反义寡核苷酸基于来自第一轮筛选的有效反义寡核苷酸，其具有长度、mRNA上的确切位置和化学修饰模式的修改。人EFO-21(卵巢囊腺癌)(图5A)、人SKOV-3(卵巢腺癌)(图5B)和小鼠4T1(乳腺癌)(图5C)细胞用10μM的相应的反义寡核苷酸不添加任何转染试剂处理3天。作为对照，用S6处理细胞，S6是一种与任何人或小鼠mRNA没有序列互补性的寡核苷酸。作为载体对照，用培养基处理细胞。在第一轮筛选中显示出有效活性的反义寡核苷酸A05008HM、A05018HM和A05028HM用作参照。对于EFO-21细胞(表9)、SKOV-3细胞(表10)和4T1细胞(表11)列出了相对于未处理的细胞(设定为1)的归一化至HPRT1的hmCD73的平均mRNA表达的值(图5A-C)。引人注目的是，EFO-21细胞中13个测试的ASO中的11个的CD73 mRNA水平降低≥80％(参见图5A)，SKOV-3细胞中的13个测试的ASO中的7个降低≥80％(参见图5B)，并且在4T1细胞中的13个测试的ASO中的9个降低≥80％(参见图5C)。用对照寡核苷酸S6处理在三种细胞系中没有有效地降低CD73mRNA。For the third round of screening, new antisense oligonucleotides were designed. These antisense oligonucleotides are based on effective antisense oligonucleotides from the first round of screening with modifications in length, exact position 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 were treated with 10 μM of the corresponding antisense oligo Nucleotides were treated for 3 days without adding any transfection reagent. As a control, cells were treated with S6, an oligonucleotide with no sequence complementarity to any human or mouse mRNA. As a vehicle control, cells were treated with medium. Antisense oligonucleotides A05008HM, A05018HM, and A05028HM that showed potent activity in the first round of screening were used as references. For EFO-21 cells (Table 9), SKOV-3 cells (Table 10) and 4T1 cells (Table 11) the mean values of hmCD73 normalized to HPRT1 are listed relative to untreated cells (set to 1). Values of mRNA expression (Fig. 5A-C). Strikingly, CD73 mRNA levels were reduced by ≥80% in 11 of 13 tested ASOs in EFO-21 cells (see Figure 5A) and in 7 of 13 tested ASOs in SKOV-3 cells ≥80% (see FIG. 5B ), and 9 out of 13 tested ASOs were reduced by ≥80% in 4T1 cells (see FIG. 5C ). Treatment with control oligonucleotide S6 did not effectively reduce CD73 mRNA in the three cell lines.
表9：相对于未处理的细胞(设定为1)，反义寡核苷酸处理的EFO-21细胞中的平均归一化hmCD73 mRNA表达值。Table 9: Mean normalized hmCD73 mRNA expression values in antisense oligonucleotide-treated EFO-21 cells relative to untreated cells (set as 1).
表10：相对于未处理的细胞(设定为1)，反义寡核苷酸处理的SKOV-3细胞中的平均归一化hmCD73 mRNA表达值。Table 10: Mean normalized hmCD73 mRNA expression values in antisense oligonucleotide-treated SKOV-3 cells relative to untreated cells (set as 1).
表11：相对于未处理的细胞(设定为1)，反义寡核苷酸处理的4T1细胞中的平均归一化hmCD73 mRNA表达值。Table 11 : Mean normalized hmCD73 mRNA expression values in antisense oligonucleotide-treated 4T1 cells relative to untreated cells (set as 1).
实施例6：在EFO-21细胞中第三轮筛选的选择的hmCD73反义寡核苷酸的IC50测定(mRNA水平)Example 6:IC50 determination (mRNA level) of selected hmCD73 antisense oligonucleotides in the third round of screening in EFO-21 cells
hmCD73反义寡核苷酸A05038HM(SEQ ID No.42)、A05041HM(SEQ ID No.45)、A05042HM(SEQ ID No.45)和A05044HM(SEQ ID No.46)在三种细胞系EFO-21、SKOV-3和4T1中显示出有效的单剂量活性。为了研究效果的浓度依赖性并为了测定IC50值，用10μM、3.3μM、1.1μM、370nM、120nM、41nM、14nM或4.5nM的相应的反义寡核苷酸处理EFO-21-细胞。在第一轮筛选中显示出有效活性的反义寡核苷酸A05018HM用作参照。处理3天后分析hCD73mRNA表达。图6描绘了hmCD73反义寡核苷酸对hCD73表达的浓度依赖性降低。GraphPadPrism计算的IC50值如表12所示：hmCD73 antisense oligonucleotides A05038HM (SEQ ID No.42), A05041HM (SEQ ID No.45), A05042HM (SEQ ID No.45) and A05044HM (SEQ ID No.46) in three cell lines EFO-21 , SKOV-3 and 4T1 showed potent single-dose activity. To study the concentration dependence of the effect and to determineIC50 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 the corresponding antisense oligonucleotides. Antisense oligonucleotide A05018HM, which showed potent activity in the first round of screening, was used as a reference. hCD73 mRNA expression was analyzed 3 days after treatment. Figure 6 depicts the concentration-dependent reduction of hCD73 expression by hmCD73 antisense oligonucleotides. TheIC50 values calculated by GraphPadPrism are shown in Table 12:
表12：在EFO-21细胞中在滴定浓度下选择的hmCD73反义寡核苷酸的IC50值和靶标抑制Table 12:IC50 values and target inhibition of selected hmCD73 antisense oligonucleotides at titrated concentrations in EFO-21 cells
实施例7：A05008HM(SEQ ID No.4)、A05018HM(SEQ ID No.3)和A05028HM(SEQ IDNo.5)对CD73蛋白表达和细胞活力的浓度依赖性影响Example 7: Concentration-dependent effects of A05008HM (SEQ ID No.4), A05018HM (SEQ ID No.3) and A05028HM (SEQ ID No.5) on CD73 protein expression and cell viability
高效hmCD73反义寡核苷酸A05008HM(SEQ ID No.4)、A05018HM(SEQ ID No.3)和A05028HM(SEQ ID No.5)关于它们对hCD73蛋白表达的敲除效力及它们在不同浓度下对细胞活力的影响详细表征。因此，用不同浓度的相应的反义寡核苷酸处理SKOV-3细胞三天。然后，将细胞在含有指定浓度的反义寡核苷酸的新鲜DMEM培养基中温育另外3天。使用CD73抗体(克隆AD2)和7-AAD通过流式细胞术分析蛋白质表达以研究活力。如图7A所示，所有三种反义寡核苷酸在所有指定的浓度下显示出hCD73蛋白的有效抑制，而用neg1的处理没有抑制作用。然而，细胞活力部分地受A05028HM影响(图7B)。相反，A05008HM(SEQ ID No.4)和A05018HM(SEQ ID No.3)在任何测试的条件下都不影响SKOV-3细胞的活力。表13总结了SKOV-3细胞中选择的人CD73反义寡核苷酸A05008HM(SEQ ID No.4)、A05018HM(SEQ IDNo.3)和A05028HM(SEQ ID No.5)的蛋白质敲除效率：High-efficiency hmCD73 antisense oligonucleotides A05008HM (SEQ ID No.4), A05018HM (SEQ ID No.3) and A05028HM (SEQ ID No.5) about their hCD73 protein expression knockout efficacy and their under different concentrations The effect on cell viability was characterized in detail. Therefore, SKOV-3 cells were treated with different concentrations of the corresponding antisense oligonucleotides for three days. Cells were then incubated for an additional 3 days in fresh DMEM medium containing the indicated concentrations of antisense oligonucleotides. Viability was studied by analyzing protein expression by flow cytometry using CD73 antibody (clone AD2) and 7-AAD. As shown in Figure 7A, all three antisense oligonucleotides showed potent inhibition of hCD73 protein at all indicated concentrations, whereas 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 the viability of SKOV-3 cells under any of the conditions tested. Table 13 summarizes the protein knockout efficiency of human CD73 antisense oligonucleotides A05008HM (SEQ ID No.4), A05018HM (SEQ ID No.3) and A05028HM (SEQ ID No.5) selected in SKOV-3 cells:
表13：在SKOV-3细胞中在滴定浓度下选择的hmCD73反义寡核苷酸的CD73蛋白表达的抑制Table 13: Inhibition of CD73 protein expression by selected hmCD73 antisense oligonucleotides at titrated concentrations in SKOV-3 cells
实施例8：CD73蛋白敲除对人EFO-21和SKOV-3细胞中的细胞外焦磷酸酯水平的影响Example 8: Effect of CD73 protein knockout on extracellular pyrophosphate levels in human EFO-21 and SKOV-3 cells
腺苷是通过hmCD73的ATP降解过程中产生的一种主要的免疫抑制分子。可以通过比色磷酸酯测定试剂盒(ab65622，abcam)通过在AMP降解为腺苷期间产生的焦磷酸酯间接检测腺苷。因此，用5μM的反义寡核苷酸A05018HM处理人SKOV-3和EFO21细胞6天(3+3)。在3天后，用含有5μM的反义寡核苷酸的新鲜DMEM培养基替换DMEM培养基。通过流式细胞术在6天后确认两种测试的细胞系中的CD73的蛋白质敲除(图8)。作为对照，用neg1处理细胞(图8)。在指定的时间点向细胞中加入500μM的AMP后，在细胞上清液中分析EFO-21细胞(图9A)和SKOV-3细胞(图9C)产生的游离磷酸酯。如由细胞滴度蓝(cell titer blue)测试研究的，用反义寡核苷酸的处理不影响EFO-21细胞(图9B)和SKOV-3细胞(图9D)的细胞活力。值得注意的是，用A05018HM处理后，EFO-21和SKOV-3细胞的磷酸酯生产效率明显降低(图9A，9C)，导致与用阴性对照neg1处理的细胞和与未处理的细胞相比，磷酸酯浓度低约1.5倍(图9A、9C、表14)。因此，抗CD73特异性反义寡核苷酸处理可以有效阻断癌细胞的腺苷产生。在表14中，给出了来自用A05018HM处理的SKOV-3和EFO21细胞的细胞上清液的磷酸酯浓度以及来自相应的对照反应的浓度。Adenosine is a major immunosuppressive molecule produced during ATP degradation by hmCD73. Adenosine can be detected indirectly through the pyrophosphate produced during the degradation of AMP to adenosine by a colorimetric phosphate assay kit (ab65622, abcam). 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 of antisense oligonucleotide. Protein knockdown of CD73 in both tested cell lines was confirmed by flow cytometry after 6 days (Figure 8). As a control, cells were treated with neg1 (Fig. 8). Free phosphate produced by EFO-21 cells (FIG. 9A) and SKOV-3 cells (FIG. 9C) was analyzed in cell supernatants after addition of 500 [mu]M AMP to the cells at the indicated time points. Treatment with antisense oligonucleotides did not affect cell viability of EFO-21 cells ( FIG. 9B ) and SKOV-3 cells ( FIG. 9D ), as investigated by the cell titer blue assay. Notably, the phosphate production efficiency of EFO-21 and SKOV-3 cells was significantly reduced after treatment with A05018HM (Fig. 9A, 9C), resulting in an Phosphate concentrations were about 1.5-fold lower (Fig. 9A, 9C, Table 14). Therefore, anti-CD73-specific antisense oligonucleotide treatment can effectively block the adenosine production of cancer cells. In Table 14, the phosphate concentrations of cell supernatants from SKOV-3 and EFO21 cells treated with A05018HM and the concentrations from the corresponding control reactions are given.
表14：在CD73蛋白敲除后和向细胞添加外源AMP后，作为人EFO-21和SKOV-3细胞上清液中的腺苷的间接检测方法的磷酸酯浓度测定。Table 14: Phosphate concentration determination as an indirect method of detection of adenosine in human EFO-21 and SKOV-3 cell supernatants following CD73 protein knockdown and addition of exogenous AMP to cells.
实施例9：hmCD73特异性反义寡核苷酸对人卵巢癌细胞系的细胞外AMP的降解和至腺苷的转化的影响的研究Example 9: Study of the effect of hmCD73-specific antisense oligonucleotides on the degradation of extracellular AMP and conversion to adenosine in human ovarian cancer cell lines
A05018HM在抑制人癌细胞中的hCD73 mRNA和蛋白表达方面表现出显着活性。在以下实验中，在EFO-21细胞中研究了hmCD73反义寡核苷酸处理对细胞外AMP降解为免疫抑制性腺苷的能力的影响。A05018HM exhibits significant activity in inhibiting hCD73 mRNA and protein expression in human cancer cells. In the following experiments, the effect of hmCD73 antisense oligonucleotide treatment on the ability of extracellular AMP to degrade into immunosuppressive adenosine was investigated in EFO-21 cells.
AMP以及腺苷水平可以通过质谱测定。为了分析hmCD73反义寡核苷酸处理对人EFO-21细胞将细胞外AMP转化为腺苷的能力的影响，用5μM的反义寡核苷酸A05018HM或对照寡核苷酸S6处理细胞，总处理时间为6天。作为载体对照，仅用培养基处理细胞。AMP and 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 into adenosine, cells were treated with 5 μM of antisense oligonucleotide A05018HM or control oligonucleotide S6, total Processing time is 6 days. As a vehicle control, cells were treated with medium only.
在处理6天后通过流式细胞术分析CD73蛋白表达，并且与用S6处理的细胞(白色柱；图10A)相比，在用A05018HM处理的细胞(黑色柱)中被显着抑制。为了研究降解细胞外AMP的能力，在处理6天后，用补充有300μM的AMP的PBS替换细胞培养基，并温育6.5小时。为了研究在不存在细胞的情况下AMP的降解，将300μM的AMP加入到无细胞的PBS中并孵化6.5h(斑点柱)。CD73 protein expression was analyzed by flow cytometry after 6 days of treatment and was significantly suppressed in cells treated with A05018HM (black bars) compared to cells treated with S6 (white bars; Figure 10A). To investigate the ability to degrade extracellular AMP, after 6 days of treatment, the cell culture medium was replaced with PBS supplemented with 300 μM of AMP and incubated for 6.5 hours. To study the degradation of AMP in the absence of cells, 300 μM of AMP was added to cell-free PBS and incubated for 6.5 h (spotted column).
此后，通过质谱测定细胞上清液(细胞条件PBS)和无细胞PBS中的AMP和腺苷水平。与无细胞PBS中的AMP水平(条带柱；图10B)相比，未处理的细胞的细胞上清液中的AMP水平(虚线柱)有效降低，而腺苷水平增加(图10C)。这表明细胞相关的AMP至腺苷的转化能力强。引人注目的是，在与S6处理的细胞(白色柱)或载体处理的细胞的上清液(斑点柱；图10B和10C，表15和16)相比时，在A05018HM处理的EFO-21细胞(黑色柱)的上清液中AMP水平显着增加，且腺苷水平显着降低。因此，这些数据清楚地表明用A05018HM处理人EFO-21细胞显着抑制细胞将细胞外AMP转化为免疫抑制性腺苷的能力。Thereafter, AMP and adenosine levels in cell supernatants (cell-conditioned PBS) and cell-free PBS were determined by mass spectrometry. Compared to AMP levels in cell-free PBS (striped columns; FIG. 10B ), AMP levels in cell supernatants of untreated cells (dashed columns) were effectively decreased, while adenosine levels were increased ( FIG. 10C ). This indicates a strong conversion of cell-associated AMP to adenosine. Strikingly, when compared to supernatants of S6-treated cells (white bars) or vehicle-treated cells (spotted bars; Figures 10B and 10C, Tables 15 and 16), EFO-21 treated with A05018HM AMP levels were significantly increased and adenosine levels were significantly decreased in the supernatant of cells (black bars). Thus, these data clearly demonstrate that treatment of human EFO-21 cells with A05018HM significantly inhibits the cells' ability to convert extracellular AMP into immunosuppressive adenosine.
表15：用AMP处理EFO-21细胞后相对于无细胞PBS(设定为1)的细胞上清液中的相对AMP浓度的测定。Table 15: Determination of relative AMP concentrations in cell supernatants after treatment of EFO-21 cells with AMP relative to cell-free PBS (set as 1).
表16：用AMP处理EFO-21细胞后细胞上清液中的绝对腺苷浓度的测定。Table 16: Determination of absolute adenosine concentrations in cell supernatants after treatment of EFO-21 cells with AMP.
实施例10：在存在或不存在细胞外ATP的情况下hmCD73特异性反义寡核苷酸对T细胞增殖的影响的研究Example 10: Study of the effect of hmCD73-specific antisense oligonucleotides on T cell proliferation in the presence or absence of extracellular ATP
本发明的先前结果(参见实施例9)揭示了用A05018HM处理人癌细胞系显着抑制了它们将细胞外AMP转化为腺苷的能力。由于CD39-CD73轴对T细胞功能起重要作用，因此研究了在存在或不存在细胞外AMP情况下，A05018HM对人CD4+T细胞的增殖的影响。因此，用细胞增殖染料标记人CD4+T细胞，用抗CD3激活并用5μM的反义寡核苷酸A05018HM(黑色柱)或对照寡核苷酸S6(白色柱)处理5天的总处理时间。在载体对照(条纹柱)中，仅用抗CD3激活细胞。随后，在寡核苷酸处理开始后第3天和第4天将300μM的AMP或载体加入细胞中。在寡核苷酸处理开始后第5天，使用流式细胞术分析CD4+T细胞的CD73蛋白表达、增殖和绝对细胞数。Previous results of the present invention (see Example 9) revealed that treatment of human cancer cell lines with A05018HM significantly inhibited their ability to convert extracellular AMP to adenosine. Since the CD39-CD73 axis is important for T cell function, the effect of A05018HM on the proliferation of human CD4+ T cells in the presence or absence of extracellular AMP was investigated. Accordingly, human CD4+ T cells were labeled with a cell proliferation dye, activated with anti-CD3 and treated with 5 μΜ of antisense oligonucleotide A05018HM (black bars) or control oligonucleotide S6 (white bars) for a total treatment time of 5 days. In vehicle controls (striped columns), cells were activated with anti-CD3 only. Subsequently, 300 μM of AMP or vehicle was added to the cells on days 3 and 4 after the initiation 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.
CD4+T细胞的A05018HM处理有效抑制CD73蛋白表达(图11A)。在没有细胞外AMP的情况下，在A05018HM、S6、和载体处理的CD4+T细胞之间未观察到增殖(图11B上图)或绝对细胞数(图11C)的差异。引人注目的是，补充300μM的AMP减少增殖(图11B下图)并且显着降低用S6或载体处理的CD4+T细胞的绝对数量(图11C)。相反，通过补充含有AMP的细胞培养基，没有减少A05018HM处理的CD4+T细胞的增殖(图11B下图)。因此，在A05018HM处理的细胞中，AMP补充几乎不减少绝对T细胞数(图11C)。A05018HM treatment of CD4+ T cells effectively suppressed CD73 protein expression (Fig. 11A). In the absence of extracellular AMP, no differences in proliferation (FIG. 11B upper panel) or absolute cell numbers (FIG. 11C ) were observed between A05018HM, S6, and vehicle-treated CD4+ T cells. Strikingly, supplementation with 300 μΜ AMP reduced proliferation (Fig. 11B lower panel) and significantly reduced the absolute number of CD4+ T cells treated with S6 or vehicle (Fig. 11C). In contrast, the proliferation of A05018HM-treated CD4+ T cells was not reduced by supplementing the cell culture medium with AMP (Fig. 1 IB lower panel). Thus, AMP supplementation hardly reduced absolute T cell numbers in A05018HM-treated cells (Fig. 11C).
总之，这些结果表明，补充含有AMP的细胞培养基显着减少了表达CD4+T细胞的CD73的增殖和绝对细胞数。引人注目的是，通过A05018HM处理的CD73蛋白敲除逆转了补充的AMP对T细胞增殖的抑制作用，最可能通过抑制免疫抑制性腺苷的形成。Taken together, these results demonstrate that supplementation of cell culture media with AMP significantly reduces the proliferation and absolute cell numbers of CD73-expressing CD4+ T cells. Strikingly, knockdown of CD73 protein by A05018HM treatment reversed the inhibitory effect of supplemented AMP on T cell proliferation, most likely by inhibiting the formation of immunosuppressive adenosine.
实施例11：人/小鼠交叉反应CD73反义寡核苷酸(A05027HM)在小鼠肝脏中的体内mCD73 mRNA敲除Example 11: In vivo mCD73 mRNA knockout in mouse liver by human/mouse cross-reactive CD73 antisense oligonucleotide (A05027HM)
选择有效的hmCD73反义寡核苷酸A5027HM，并研究在全身给予未配制的寡核苷酸后其对Balb/c小鼠的肝脏中的mCD73 mRNA表达的影响。因此，在第1、2、3、4、5、8、10和12天以20mg/kg的剂量皮下注射A05027HM处理Balb/c小鼠(5只小鼠/组)。作为对照，用载体处理Balb/c小鼠(盐水，6只小鼠/组)。在最后一次反义寡核苷酸处理(第15天)后三天，处死小鼠并取样肝脏用于分析mCD73 mRNA水平。图12中描绘的结果显示A05027HM或载体处理的小鼠(白色柱)的肝脏中的mCD73 mRNA水平。引人注目的是，与载体对照相比，在用A05027HM处理小鼠后mCD73 mRNA水平显着(p＝0.0043)减少。A potent hmCD73 antisense oligonucleotide, A5027HM, was selected and its effect on mCD73 mRNA expression in the liver of Balb/c mice following systemic administration of the unformulated oligonucleotide was studied. Therefore, Balb/c mice (5 mice/group) were treated with subcutaneous injection of A05027HM at a dose of 20 mg/kg on days 1, 2, 3, 4, 5, 8, 10 and 12. As a control, Balb/c mice (saline, 6 mice/group) were treated with vehicle. Three days after the last antisense oligonucleotide treatment (day 15), mice were sacrificed and livers were sampled for analysis of mCD73 mRNA levels. Results depicted in Figure 12 show mCD73 mRNA levels in livers of A05027HM or vehicle treated mice (white bars). Strikingly, mCD73 mRNA levels were significantly (p=0.0043) reduced after treatment of mice with A05027HM compared to vehicle controls.
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